Mammalian Cytochrome P450 Enzymes Catalyze thePhenol-coupling Step in Endogenous Morphine Biosynthesis*□S

Received for publication, April 21, 2009, and in revised form, May 27, 2009 Published, JBC Papers in Press, June 26, 2009, DOI 10.1074/jbc.M109.011320

Nadja Grobe‡, Baichen Zhang‡, Ursula Fisinger§, Toni M. Kutchan‡, Meinhart H. Zenk‡1, and F. Peter Guengerich¶

From the ‡Donald Danforth Plant Science Center, St. Louis, Missouri 63132, the §Lehrstuhl fur Pharmazeutische Biologie,Universitat Munchen, Karlstrasse 29, 80333 Munchen, Germany, and the ¶Department of Biochemistry and Center in MolecularToxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146

A cytochrome P450 (P450) enzyme in porcine liver that cata-lyzed the phenol-coupling reaction of the substrate (R)-reticu-line to salutaridine was previously purified to homogeneity(Amann, T., Roos, P. H., Huh, H., and Zenk, M. H. (1995)Hetero-cycles 40, 425–440). This reaction was found to be catalyzed byhuman P450s 2D6 and 3A4 in the presence of (R)-reticuline andNADPH to yield not a single product, but rather (�)-isoboldine,(�)-corytuberine, (�)-pallidine, and salutaridine, the para-ortho coupled established precursor of morphine in the poppyplant and most likely also in mammals. (S)-Reticuline, a sub-strate of both P450 enzymes, yielded the phenol-coupled alka-loids (�)-isoboldine, (�)-corytuberine, (�)-pallidine, andsinoacutine; none of these serve as a morphine precursor. Cata-lytic efficiencies were similar for P450 2D6 and P450 3A4 in thepresence of cytochrome b5 with (R)-reticuline as substrate. Themechanism of phenol coupling is not yet established; however,we favor a single cycle of iron oxidation to yield salutaridine andthe three other alkaloids from (R)-reticuline. The total yield ofsalutaridine formed can supply the 10 nM concentration ofmor-phine found in human neuroblastoma cell cultures and in braintissues of mice.

Cytochrome P450 (P450)2 enzymes catalyze the most versa-tile chemical reactions in nature (1). There is, however, a dis-crepancy between the plant and the animal kingdoms withregard to the sheer number of these biocatalysts. Whereas in asingle model plant, Arabidopsis thaliana, there are to date 273P450 proteins, in the human genome, only 57 of these proteinsare present. Whereas plants and animals share a multitude ofhighly regio- and stereospecific O-demethylation reactions,more complex reactions such as phenol coupling are muchmore abundant in plants than in animals, especially in the alka-loid field (2–11). The proposal of Barton and Cohen (12) cor-related the structure of specific plant alkaloids in terms of thisreaction mechanism and gave mechanistic proposals of how

these phenol-coupled products may possibly be biosynthesizedin nature. The oxidation of phenols by one-electron transferaffords radicals, which, by radical pairing, form new C-C orC-Obonds either by intra- or intermolecular coupling. The firsttwo examples that unequivocally demonstrated the formationof C-C andC-Obonds in a stereo- and regioselectivemanner inplant metabolism are catalyzed by specific P450-linked micro-somal-bound plant enzymes (13). One of these enzymes wassalutaridine synthase fromPapaver somniferum (opiumpoppy)(14). This synthase catalyzes the intramolecular formation ofthe critical C12-C13 carbon bridge and is a key enzyme inmor-phine biosynthesis.The groups of Goldstein (15) and Spector (16) have pub-

lished a number of reports over the past 25 years claiming thatmammals are capable of synthesizing de novo traces of endog-enous morphine. However, no convincing experimental datahave been presented regarding the enzymes. Phenol-couplingreactions inmammals are extremely rare, and the only exampledescribed thus far is the formation of thyroxine, by radical pair-ing, in humans. If the key step of morphine synthesis, the for-mation of phenol-coupled salutaridine from (R)-reticuline,occurs in mammals then in analogy to plants, a P450 enzymemust be present (inmammals) to catalyze this reaction. In 1987,the first experiments were conducted in an attempt to discoverthe reaction by supplying uniformly labeled racemic [3H]reti-culine in the presence of rat microsomes andNADPH to exam-ine whether [3H]salutaridine can be formed under these condi-tions (15). A radioactive compound was formed in 1% yield andassumed to be the phenol-coupled product salutaridine. Welater repeated this experiment using (R)-[N-14CH3]reticulineand NADPH-fortified microsomes from pig, rat, cow, andsheep. We observed the formation of [N-14CH3]salutaridinewith the correct stereochemistry at carbon 9 (17). The enzymefrom porcine liver was subsequently purified to homogeneityand the reaction product was characterized by mass spectrom-etry and physical parameters to be a product of a P450 enzyme,whichwe provisionally named “salutaridine synthase” (18). Theaim of this report is the identification of the homogenous por-cine P450 enzyme, its equivalent in humans, and the mecha-nism of the phenol-coupling reaction in the formation of pre-cursors of morphine.

EXPERIMENTAL PROCEDURES

Chemicals—Alkaloids were from our departmental collec-tion, and isoboldine, pallidine, and sinoacutine were gifts of Dr.Andre Cave, University Paris-Sud, Dr. Shoei-Sheng Lee,

* This work was supported, in whole or in part, by National Institutes ofHealth Grants R21 DA0224418 and R37 CA090426 and the DeutscheForschungsgemeinschaft.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. S1–S7.

1 To whom correspondence should be addressed: Donald Danforth Plant Sci-ence Center, 975 North Warson Rd., St. Louis, MO 63132. Tel.: 314-587-1474; Fax: 314-587-1574; E-mail: mhzenk@danforthcenter.org.

2 The abbreviations used are: P450, cytochrome P450; EPI, enhanced production; LC, liquid chromatography; MRM, multiple reaction monitoring; MS,mass spectrometry; TLC, thin layer chromatography.

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National Taiwan University, and Dr. F. Bracher, Ludwig-Maxi-milians-University Munich. L-�-Dilauroyl-sn-glycero-3-phos-phocholine, bovine liver catalase, and bovine erythrocytessuperoxide dismutase were obtained from Sigma.Enzymes—Human P450 2D6 and rat NADPH-P450 reduc-

tase were prepared as described (19). Human P450 2D6 andhuman P450 3A4 (with or without cytochrome b5) were pur-chased from BD Biosciences (Woburn, MA). Porcine salutari-dine synthasewas purified according to Ref. 18 and subjected toSDS-PAGE on a 10% (w/v) acrylamide gel. The protein band at�50 kDa (containing �10 �g of protein) was excised andeluted, and the N terminus was sequenced by Edman degrada-tion using an Applied Biosystems Model 470 gas-phasesequencer at the Max Planck Institute for Biochemistry, Mar-tinsried, Germany.Enzyme Assays—Enzyme assays utilizing radioactive labeled

substratewere conducted in a total volume of 140�l containing36 mM potassium phosphate buffer (pH 6.5), 20 pmol of P450enzyme (BD Biosciences, microsomal preparation from bacu-lovirus-infected insect cells coexpressing human NADPH-P450 reductase), 1.8 mM NADPH, 0.36 mM EDTA, and 0.2 �Ci(R)- or (S)-[N-14CH3] reticuline (200,000–300,000 cpm, 6.75nmol). The reactionmixture was incubated for 5 h at 37 °C. Foridentification of products, the reaction mixture was separatedby two-dimensional TLC (solvent 1: toluene/acetone/ethanol/NH4OH, 45:45:7:3 (v/v/v/v); solvent 2: CHCl3/acetone/diethyl-amine, 5:4:1 (v/v/v)), and radioactive bands were detected byphosphorimagingwith a Typhoon 9410 (Molecular Dynamics).For plant-feeding experiments, potential precursor solutionswere obtained by separation of the reaction mixture by TLCusing the solvent mixture chloroform/acetone/diethylamine,5:4:1 (v/v/v) following the elution of corresponding bands withCH3OH and reconstitution in H2O. Standard reactions forkinetic data and pH profile analysis were conducted in a totalvolume of 250 �l containing 100 mM potassium phosphatebuffer (pH 7.4), 15 �g of L-�-dilauroyl-sn-glycero-3-phospho-choline, 1 mM NADP�, 10 mM glucose 6-phosphate, 1 unit/mlglucose-6-phosphate dehydrogenase (yeast), 1000 units/ml cat-alase (bovine liver), 10 �g bovine erythrocyte superoxide dis-mutase, and 0.25 �M to 5 mM substrate. Reactions were startedby adding 38 pmol of P450 2D6 (Escherichia coli-expressed pro-tein) and 255 pmol of rat cytochrome P450 reductase (E. coli-expressed protein) or 9–18 pmol of P450 2D6 or P450 3A4 (BDBiosciences) and incubated for 10 min at 37 °C. Reaction mix-tures were made alkaline with 400 �l of 1 MNa2CO3 buffer (pH9.5) and extracted twice with 400 �l of CHCl3. The combinedorganic phases were dried, reconstituted with CH3OH, andsubjected to LC-MS/MS. Kinetic parameters were estimated bynon-linear regression with GraphPad Prism. For pH profileanalysis enzymatic assayswere conducted as described above ina reaction mixture containing 25 �mol of following buffer:sodium citrate (pH 4–6), potassium phosphate (pH 6–8), andTris/HCl (pH 8–9).Plant Feeding Experiments—Sterilized 5-day-old Papaver

seedlings were incubated for 48 h with the potential precursorsolution. The seedlings were extracted with 80% ethanol (v/v).The extracts were dried, redissolved in 20 �l 50% ethanol (v/v),and separated by TLC in solvent system toluene/ethyl acetate/

diethylamine, 7:2:1 (v/v/v). Radioactive bands were detected byphosphorimaging.LC-MS/MS Analysis—Enzyme activities were analyzed with

a 4000 QTRAP LC-TIS-MS-MS system by monitoring theenhanced product ion (EPI) and multiple reaction monitoring(MRM) in the positive ionization mode. The system consistedof a CTC PAL autosampler (LEAP Technologies), a ShimadzuLC-20AD HPLC system, and a 4000 QTRAP mass spectrome-ter (Applied Biosystems). Separation of (10 �l) samples wasachieved by using a Luna C18 octadecylsilane HPLC column(Phenomenex, 5 �m, 150 mm � 2 mm) combined with a C18guard column (Phenomenex, 4mm� 2mm). Themobile phasetotal flow was set to 0.5 ml/min with binary gradient elution,using solvent A (5%CH3OH, 5%CH3CN, 10mMNH4HCO3, 45mM NH4OH) and B (90% CH3CN, 10 mM NH4HCO3, 15 mM

NH4OH) (all v/v). The gradient started with 100% A for 2 minand was increased to 100% B over 10 min. Elution was contin-ued for 2 min at 100% B followed by a 5-min equilibration withstarting condition. The following TIS source parameters wereused: CUR 30, CAD high, IS 5000, TEM 500, EP 10, CXP 17.Compound-dependent parameters for the compounds of inter-est (collision energy, declustering potential, quantifier MRMtransition, qualifier MRM transition, dwell time) are listed inTable 1. Identification of an analyte was based on retentiontimes, the quantifier-to-qualifier MRM transition ratio, andcomparison with the expected values for standards. Quantita-tion was performed by constructing standard curves for eachanalyte and integrating the peak area of the quantifier MRMtransition with Analyst 1.4.1 (Applied Biosystems,MDS SCIEXInstruments).

RESULTS

Sequencing of Porcine Salutaridine Synthase—The microso-mal porcine liver enzyme catalyzing the formation of the phe-nol-coupling reaction from (R)-reticuline to salutaridine waspurified to apparent electrophoretic homogeneity and sub-jected to Edman degradation. The N terminus yielded theamino acid sequence: Gly-Leu-Leu-Thr-Gly-Asp-Leu-Leu-Gly-Ile-Leu-Ala-Leu-Ala-Met-Val-Ile-Phe-Leu-Leu-Asn-(Leu)-Val-Asp-Leu-Met-X-Arg.Upon comparison of this peptide sequence to those present

in the public databases, this is the exact N-terminal of pig P4502D25, except for the missing N-terminal Met. Homology tohuman P450 2D6 was observed (56% identity). Consequently,human P450 2D6 was obtained from a commercial source (BDBiosciences) and found to indeed catalyze the phenol-coupling

TABLE 1Compound-dependent parameters for the LC-MS-MS method

Analyte Collisionenergy

Declusteringpotential

QuantifierMRM

transition

QualifierMRM

transitionDwelltime

V V msReticuline 35 45 3303 192 3303 137 50Corytuberine 40 50 3283 265 3283 282 50Pallidine 40 50 3283 211 3283 237 50Salutaridine 40 50 3283 211 3283 237 50Isoboldine 40 50 3283 265 3283 237 50Thebaine 35 40 3123 251 3123 281 50Oripavine 35 40 2983 218 2983 249 50

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reaction from (R)-[N-14CH3]reti-culine to salutaridine in the pres-ence of NADPH. However, closeexamination of the reaction prod-ucts of the porcine and the commer-cially available human P450 2D6enzymes by two-dimensional TLCrevealed four metabolites in addi-tion to unconsumed 14C-labeled(R)-reticuline (vide infra).Identification of P450 2D6-cata-

lyzed Reaction Products—Incuba-tion of (R)-reticuline in the presenceof NADPH-P450 reductase andP450 2D6 resulted in the formationof four metabolites that were notfound in controls with heat-inacti-vated enzymes. These metaboliteswere subjected to MS after separa-tion by HPLC. Comparison of thefour compounds with standards ofalkaloids that were expected to beformed according to the phenol-coupling reactions allowed theidentification of the unknown com-pounds as salutaridine (para-orthocoupling), (�)-pallidine (para-paracoupling), (�)-isoboldine (ortho-para coupling), and (�)-corytuber-

ine (ortho-ortho coupling) (Fig. 1). The identification of thesecompounds is consistentwith the previous findings by the incu-bation of rat liver microsomes in the presence of racemic reti-culine (20, 21). However, the important morphine precursorsalutaridine was missed during that investigation. Salutaridineis formed unequivocally during the phenol-coupling reactionandwas identified byMS and retention time. Application of thepara-ortho coupled product to 5-day-old poppy seedlings ver-ified salutaridine by its incorporation into thebaine (Fig. 2),while the other three phenol-coupled products of the (R)-serieswere not incorporated into thebaine. Analogous incubation ofNADPH-P450 reductase in the presence of P450 2D6 and (S)-reticuline again gave four phenol-coupled products of the (S)-series, which were sinoacutine, (�)-pallidine, (�)-isoboldine,and (�)-corytuberine and did not serve as precursors to mor-phine. The collision-induced dissociation (CID) spectra of salu-taridine [M�H]� 328 by EPI yielded the characteristic frag-ment ions m/z 297, 285, 265, 239, and 237 (22) that were alsofound in CID spectra of the para-ortho coupled product. TheCID spectra of salutaridine standard and para-ortho coupledenzymatic product are shown (Fig. 3).Human P450 2D6 is known to catalyze a multitude of reac-

tions both with natural compounds and xenobiotics and isinvolved in the oxidation of about 30% of drugs used by humans(19, 23). Interestingly, human P450 2D6 is known to catalyzeseveral reactionswithin the putative pathway leading from L-ty-rosine to morphine. These reactions include the hydroxylationof tyramine to dopamine (23, 24), the demethylation of the-baine to oripavine (25), and the demethylation of codeine to

FIGURE 1. Oxidative phenol-coupling reaction of (R)-reticuline in mammals. The four phenol-coupledproducts salutaridine, (�)-pallidine, (�)-isoboldine, and (�)-corytuberine were formed from (R)-reticuline viaan oxidative phenol-coupling reaction catalyzed by human P450 2D6 and P450 3A4.

FIGURE 2. TLC radiogram of feeding of mammalian phenol-coupledalkaloids to P. somniferum seedlings. A, 14C-labeled reticuline standard.B, 14C-labeled salutaridine formed by enzymatic phenol-coupling of (R)-[N-14CH3]reticuline. C, 14C-labeled thebaine in extract of Papaver seedlingsfed with 14C-labeled salutaridine shown in B. D, 14C-labeled sinoacutineformed by enzymatic phenol-coupling of (S)-[N-14CH3]reticuline; E, extractof Papaver seedlings fed with 14C-labeled sinoacutine shown in D. Sinoa-cutine was not incorporated into thebaine, an intermediate of plant mor-phine biosynthesis. the, thebaine; sal, salutaridine; sin, sinoacutine; ret,reticuline.

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morphine (26); all of these reactions can be catalyzed by a singleenzyme-P450 2D6. Tyramine hydroxylation to dopamine playsonly a minor role, if any, in the production of dopamine inhumans; however, three P450 2D6-catalyzed reactions mayplay a role in the biosynthesis of endogenous morphine.P450 3A4 has previously been shown to N-demethylate

codeine to N-norcodeine (27). This similarity of P450 3A4 in

accepting codeine as substrate prompted us to investigatewhether P450 3A4 can catalyze the phenol coupling of (R)-reticuline. IndeedP450 3A4 showed the product pattern knownfor 2D6, i.e. the formation of salutaridine, (�)-pallidine, (�)-isoboldine, and (�)-corytuberine from the precursor (R)-reti-culine as determined byMS (Fig. 4). Themorphine biosyntheticprecursor salutaridine is indeed formed by P450 3A4 as well.

FIGURE 3. CID spectra of salutaridine in EPI mode. A, MS of salutaridine standard (parent [M�H]� 328, CE � 40V); B, MS of the enzymatic product salutaridine(parent [M�H]� 328, CE � 40V).

FIGURE 4. LCMS analysis of products formed by human P450 2D6 and P450 3A4. The MRM transition from m/z 328 to m/z 237 shows product formationafter 2 h of incubation of: A, human P450 2D6 and (R)-reticuline; B, human P450 2D6 and (S)-reticuline; C, human P450 3A4 and (R)-reticuline; and D, human P4503A4 and (S)-reticuline.

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P450 3A4 was also tested to determine if it can catalyze thephenol-coupling reaction with the (S)-configuration of reticu-line as substrate. Incubation of P450 3A4 with (S)-reticulineagain yielded four products, sinoacutine, (�)-pallidine, (�)-isoboldine, and (�)-corytuberine, as determined by MS.The four phenol-coupled alkaloids formed in the presence of

human P450 2D6 and 10 �M (R)-reticuline were individuallyquantitated after an incubation time of 10 min, in which timeproduct formation is in the linear range: (�)-corytuberine (4nM), (�)-isoboldine (54 nM), (�)-pallidine (24 nM), and salu-taridine (6 nM). With the (S)-configuration of reticuline as sub-strate under the same incubation condition the yields ofphenol-coupled products were: (�)-corytuberine (1 nM), (�)-isoboldine (61 nM), (�)-pallidine (15 nM), and sinoacutine (3nM).With P450 3A4 as catalyst, the same set of phenol-coupledalkaloids yielded the following with (R)-reticuline as substrate:(�)-corytuberine (7 nM), (�)-isoboldine (30 nM), (�)-pallidine(18 nM), and salutaridine (28 nM). With (S)-reticuline as sub-strate, under the same conditions as above and P450 3A4 ascatalyst, the yields were: (�)-corytuberine (5 nM) (�)-isobol-dine (15 nM), (�)-pallidine (7 nM), and sinoacutine (15 nM).Similar yields were obtained for the (R)- and (S)-series of phe-nol-coupled products suggesting that the two different P450soxidize reticuline via radical formation without stereoselectiv-

ity. A pH profile for both P450 enzymes catalyzing the phenolcoupling of (R)-reticuline to salutaridine is shown in supple-mental Fig. S1. The relative distribution of the four phenol-coupled products was the same after 10 min and 120 min ofincubation as well as at any pH between 5 and 9.Kinetic Parameters of the Phenol Coupling Catalyzed by P450

2D6 and 3A4—Both human P450 enzymes described herein,P450 2D6 and P450 3A4, catalyze the same reaction by convert-ing (R)-reticuline to the four phenol-coupled products but withdistinct kinetics that can be derived from theirMichaelis-Men-ten parameters (Table 2 and supplemental Figs. S2-S4). P4502D6 had a lower apparent Km of 2–3 �M for the substrate (R)-reticuline as compared with P450 3A4 (Km � 500–1000 �M)whereas a higher maximum rate (kcat) for the conversion of(R)-reticuline is observed for P450 3A4. Including cytochromeb5 in the P450 3A4 system increased the rate of product forma-tion by about 6 to 7 times whereas the Km was only slightlychanged. These findings support other studies showing that theactivity of P450 3A4 can be enhanced by cytochrome b5whereas P450 2D6 is not affected (28). Catalytic efficiencies forboth enzymes, P450 2D6 and P450 3A4-utilizing (R)-reticulineas a substrate, show that P450 2D6 seems to be more efficient;however, if cytochrome b5 is added to the P450 3A4 system,catalytic efficiencies of both enzymes become comparable.Additionally, human P450 2D6 shows a higherKm value for twoother substrates, thebaine and codeine, i.e. 190–250 and 48�M,respectively (Table 2 and supplemental Fig. S5), as comparedwith (R)-reticuline. However, increased catalytic rates for thesubstrates thebaine and codeine lead to catalytic efficiencies ofsame order of magnitude for all three substrates of P450 2D6that described herein.

DISCUSSION

It has been shown that human P450 2D6 and P450 3A4 cat-alyze what appear to be radical-induced phenol-coupling reac-tions with both (R)-and (S)-reticuline, yielding four (R)-config-ured and four (S)-configured phenol-coupled products. Asshown in Fig. 5, a mechanism can be postulated that starts withthe fully activated P450 (“compound I” form), which abstracts ahydrogen atom to create a phenoxy radical. The substraterotates in juxtaposition to the P450 iron, which is now in the

“compound II” form and can stillabstract a second phenolic hydro-gen, which then leads to immediatecoupling within a single cycle ofP450 action, yielding a water mole-cule. A simple enolization of thecyclohexanedienal ring yields thefinal product salutaridine as one ofthe (R)-coupled alkaloids and themissing link in human morphinebiosynthesis. The intramolecularC-C phenol-coupling reaction of(S)-reticuline to corytuberine cata-lyzed by CYP80G2 in the plantCop-tis japonica is postulated to occur bya diradicalmechanism (7). This pos-tulate was based on a previously

FIGURE 5. Proposed mechanism of the oxidative phenol-coupling reaction in mammals. The formation ofsalutaridine from (R)-reticuline is catalyzed by P450 2D6 and P450 3A4 and passes through a single cycle of ironoxidation.

TABLE 2Catalytic parameters for human P450 2D6 and P450 3A4

Enzyme Substrate Product Km kcat kcat//Km

�M pmol/min/pmol P450

mM�1

s�1

P450 2D6 (R)-Reticuline Corytuberine 2.7 � 0.37 0.015 � 0.001 0.09Pallidine 1.8 � 0.26 0.045 � 0.002 0.42Salutaridine 2.5 � 0.21 0.011 � 0.001 0.08Isoboldine 1.9 � 0.36 0.390 � 0.026 3.42

Thebaine Oripavine 48 � 9.6 4.55 � 0.54 1.60Codeine Morphine 250a 14a 0.93

190b 6.4b 0.56P450 3A4 withcytochromeb5

(R)-Reticuline Corytuberine 384 � 40 1.3 � 0.1 0.06Pallidine 474 � 30 14.7 � 0.3 0.52Salutaridine 1961 � 181 12.3 � 0.5 0.12Isoboldine 4860 � 860 158 � 17 0.54

P450 3A4 (R)-Reticuline Corytuberine 534 � 66 0.16 � 0.14 0.005Pallidine 490 � 38 2.97 � 0.14 0.101Salutaridine 993 � 220 2.20 � 0.36 0.037Isoboldine 577 � 130 9.37 � 1.34 0.271

a Data taken from Ref. 40.b Data taken from Ref. 41.

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proposed mechanism in the first report of a plant cytochromeP450 that catalyzes a phenol-coupling reaction, berbamuninesynthase from Berberis stolonifera (2). The concept of a diradi-cal mechanism in phenol-coupling reactions finds origin in theproposal of Barton and Cohen (12) in which radical pairingfurnishes diphenyl ether or aryl-aryl bonds. A newC-C phenol-coupling reaction catalyzed by CYP121 of Mycobacterium tu-berculosis has recently been reported (29). It is proposedtherein that the intramolecular C-C coupling of the two tyrosylside chains of cYY to cyclodipeptide proceeds via activation ofthe tyrosyl residues through a radical mechanism. A diradicalmechanism can also be proposed herein; however, the mecha-nism proposed in Fig. 5 avoids the energetic instability of aformal diradical species.Salutaridine has previously escaped detection using rat liver

microsomes (20, 21), being a minor phenol-coupled alkaloid.Salutaridine is the para-ortho phenol-coupled product, whichis the biosynthetic precursor of morphine both in the poppyplant and in humans and other mammals. This finding addsconsiderable credibility to the controversial discussion aboutwhether endogenous morphine occurs in mammals. It shouldbe pointed out that the occurrence of P450 2D6, not only in livertissue but also in human brain, has been unequivocally proven(30). P450 2D6 occurs mainly in the neocortex, telencephalon,hippocampus, diencephalon, mesencephalon, cerebellum, andmyelencephalon. The second P450 that catalyzes the phenol-coupling reactions with (R)-reticuline as substrate formingagain four phenol-coupled products, including salutaridine-3A4- is found to occur in brain,mainly in pons and cervical cord(31) and in liver and small intestine (32).The presence of endogenous morphine in brain of humans

and mammals has been suggested by various researchers (16,33) and in sterile human cell cultures, e.g. neuroblastoma cellsSH-SY5Y (34–36). The possible occurrence of morphine inhuman and mammalian cells has been a matter of controversyfor more than 30 years. A resolution of this problem can comeonly by the application of state-of-the-art analytical chemistry,biochemistry, and molecular biology, with the application ofcritical controls to exclude introduction ofmorphine from foodsources to the animals and exclusion of laboratory contamina-tion with morphine.It has been suggested that human P450 2D6 might catalyze

the transformation of (R)-reticuline to salutaridine (37). In arecent report, Hawkins and Smolke (38) tested whether P4502D6might catalyze the formation of salutaridine from (R)-reti-culine in recombinant yeast. (R,S)-Norlaudanosoline was fed tothe recombinant yeast strain which produced racemic (R,S)-reticuline. When P450 2D6 was introduced into the (R,S)-reti-culine-producing yeast strain, a product was formed that hadthe MS fragment ions of m/z 297 and m/z 265 and was desig-nated salutaridine (38). The yield of the morphine precursorwas reported to be between 6 and 8%. The yield could not befurther increased (38) by available techniques.P450 2D6 (and P450 3A4) catalyze reactions that can be

interpreted as a diradical mechanism or alternatively involvinga single cycle of iron oxidation, vide supra (Fig. 5). This mech-anismwill lead to four final alkaloid products, each alkaloid in adifferent but consistent concentration. Only one of these prod-

ucts is the desired morphine precursor salutaridine, in 0.06%concentration. Use of racemic (R,S)-reticuline (via (R,S)-nor-laudanosoline in the recombinant yeast system) leads toanother four phenol-coupled products of the (S)-series, namelythe alkaloids (�)-corytuberine, (�)-isoboldine, sinoacutine,(�)-pallidine, all known natural products. This addition of the(S)-configured compounds would produce seven alkaloids inthe recombinant system; only one product (salutaridine) is themorphine precursor. All eight phenol-coupled productsderived from (R,S)-reticuline yield the fragment ions m/z 297and 265 upon mass fragmentation (supplemental Figs. S6 andS7). Eight different structures in different concentrations willresult in the recombinant yeast strain; the yield of salutaridinewill be �0.2%. Although the low and inherent concentration ofsalutaridine generated by P450 2D6 and P450 3A4 is low for abiotechnological production of morphine, it is expected to besufficient to generate the �10 nM concentration of morphine(via salutaridine) in human cells (35) and mouse cerebellum(36).The phenol-coupling reaction of (R)-reticuline to salutari-

dine is the key reaction in the biosynthesis of morphine inplants and animals (Fig. 5). The enzyme in endogenous mor-phine biosynthesis in mammals (P450 2D6) is unique (Fig. 6) inthat it catalyzes the 3-O-demethylation of codeine tomorphine(26), the 3-O-demethylation of thebaine to oripavine (25), andthe phenol coupling of (R)-reticuline to salutaridine (thiswork).Aminor reaction, the oxidation of tyramine to dopamine that isalso catalyzed by P450 2D6 (23, 24), does not seem to play a rolein endogenous morphine biosynthesis (39). These different

FIGURE 6. Human P450 2D6 is involved in three reactions in the biosyn-thesis of morphine. A, phenol coupling of (R)-reticuline to salutaridine; B, 3-O-demethylation of thebaine to oripavine; C, 3-O-demethylation of codeineto morphine.

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reaction types, which are catalyzed by one enzyme inmorphinebiosynthesis, are rather unique in nature.

Acknowledgments—We thankDr. F. Lottspeich,Max Planck Institutefor Biochemistry, Martinsried, Germany, for sequencing the porcineP450 (done on November 20, 1992) and recognizing the homology tohuman P450 2D6. We also thank Dr. Andre Cave, University Paris-Sud, Dr. Shoei-Sheng Lee, National Taiwan University, and Dr. F.Bracher, Ludwig-Maximilians-University Munich, for their generousgift of the alkaloids isoboldine, pallidine, and sinoacutine. Finally wethank Dr. Leslie Hicks and Dr. Sophie Alvarez, Donald DanforthPlant Science Center, for sequencing the commercially available P4502D6. Funding for the QTRAP LC-MS was provided by NSF-MRIGrant DBI-0521250.

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Mammalian P450s in Morphine Biosynthesis

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F. Peter GuengerichNadja Grobe, Baichen Zhang, Ursula Fisinger, Toni M. Kutchan, Meinhart H. Zenk and

Endogenous Morphine BiosynthesisMammalian Cytochrome P450 Enzymes Catalyze the Phenol-coupling Step in

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