Proc. Natl. Acad. Sci. USAVol. 90, pp. 2890-2894, April 1993Plant Biology

Purification, characterization, and cDNA cloning of anNADPH-cytochrome P450 reductase from mung bean

(cinnamic acid 4-hydroxylase/plant flavoprotein/heterologous reconstitution/steroid 17a-hydroxylase)

MANJUNATH S. SHET*, KANAGASABAPATHI SATHASIVANt, MICHAEL A. ARLOTTO*, MONA C. MEHDYt,AND RONALD W. ESTABROOK*f*Department of Biochemistry, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235; and tDepartment ofBotany, University of Texas, Austin, TX 78713

Contributed by Ronald W. Estabrook, December 17, 1992

ABSTRACT We report here the isolation and deducedamino acid sequence of the flavoprotein, NADPH-cytochromeP450 (cytochrome c) reductase (EC 1.6.2.4), associated with themicrosomal fraction of etiolated mung bean seedlings (Vignaradiata var. Berken). An 1150-fold purification of the plantreductase was achieved, and SDS/PAGE showed a predomi-nant protein band with an apparent molecular mass of -82 kDa.The purified plant NADPH-P450 reductase gave a positivereaction as a glycoprotein, exhibited a typical flavoproteinvisible absorbance spectrum, and contained almost equimolarquantities of FAD and FMN per mole of enzyme. Specificantibodies revealed the presence of unique epitopes distinguish-ing the plant and mammalian flavoproteins as demonstrated byWestern blot analyses and inhibition studies. Peptide fragmentsfrom the purified plant NADPH-P450 reductase were se-quenced, and degenerate primers were used in PCR amplifica-tion reactions. Overlapping cDNA clones were sequenced, andthe deduced amino acid sequence of the mung bean NADPH-P450 reductase was compared with equivalent enzymes frommammalian species. Although common flavin and NADPH-binding sites are recognizable, there is only ==38% amino acidsequence identity. Surprisingly, the purified mung beanNADPH-P450 reductase can substitute for purified ratNADPH-P450 reductase in the reconstitution of the mammalian P450-catalyzed 17a-hydroxylation of pregnenolone or progesterone.

Higher plants should be a rich source of undiscoveredcytochrome P450s since plants are recognized to catalyzemonooxygenation reactions involved in diverse biosyntheticpathways concerned with the formation of many plant sec-ondary metabolites [e.g., phytoalexins (1), lignins and fla-vonoids (2-4), sterols (5), and alkaloids (6), to name but afew]. Microsomal-bound P450 reactions are dependent on thetransfer of electrons from NADPH by a common flavopro-tein, NADPH-cytochrome P450 reductase (EC 1.6.2.4). Aunique characteristic of the microsomal NADPH-P450 re-ductase is the presence of noncovalently bound FAD andFMN as prosthetic groups (7).Microsomal NADPH-cytochrome c reductase activities

have been reported for a number of plants (8-10). Theenzyme has been purified from microsomes of sweet potato(11), Catharanthus roseus (12), and Helianthus tuberosus(13) and shown to function in the hydroxylation of the C-10methyl group of geraniol (12) and the p-hydroxylation ofcinnamic acid to form p-coumaric acid (13).The present study was undertaken to compare the enzymes

from plants with equivalent enzymes from mammalian andbacterial sources. Here we describe the purification andcharacterization of an NADPH-P450 reductase prepared

from microsomes of etiolated mung bean seedlings (Vignaradiata). The amino acid sequence, deduced from nucleotidesequences of cDNA clones§, shows that the mung beanNADPH-P450 reductase shares -38% amino acid sequenceidentity with similar NADPH-P450 reductases present inmammalian tissues. Of interest is the presence in the mungbean NADPH-P450 reductase of amino acid sequences com-mon with those of orthologous mammalian enzymes andproposed as binding sites for FMN, FAD, and NADPH (14).The purified plant NADPH-P450 reductase is able to functionefficiently for electron transfer in the reconstitution of the17a-hydroxylase and 17,20-lyase activities of mammalianP450s with membrane fractions from Escherichia coli ex-pressing mammalian recombinant P450 17A.

MATERIALS AND METHODSFMN, FAD, SDS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), cytochrome c, 2-mercaptoeth-anol (ME), adenosine 2'-monophosphate (2'-AMP), and soy-bean trypsin inhibitor were purchased from Sigma. Trypsin(L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated)was obtained from Worthington. Adenosine 2',5'-diphosphate(2',5'-ADP)-Sepharose 4B was obtained from Pharmacia.Polyclar AT (polyvinylpyrrolidone) was a gift from GAFChemicals (Wayne, NJ). DE-52 anion-exchange resin andNADPH were purchased from Whatman and BoehringerMannheim, respectively. [3-14C]Cinnamic acid (51 mCi/mmol; 1 Ci = 37 GBq) was obtained from Cen Saclay (Orsay,France). Emulgen 911 was a generous gift from Kao Chemical(Tokyo). Ultrogel AcA 34 was purchased from LKB.

Etiolated mung bean seedlings (V. radiata var. Berken)were purchased locally from Calco (Dallas). The seedlingswere grown in the dark at 28-30°C and harvested after 4 days.All subsequent operations were carried out at 4°C.

Preparation of Microsomes. Fresh seedlings were homog-enized with a Polytron using 2 ml of buffer A [100 mMTris HCl (pH 7.4) containing 250 mM sucrose, 2.8 mM ME,1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride]supplemented with 2% (wt/vol) insoluble Polyclar AT per gwet weight. The homogenate was filtered through nylon clothand centrifuged at 25,000 x g for 20 min. The microsomalfraction was sedimented by centrifugation at 100,000 x g for60 min or by vesicularization using 50 mM MgCl2 for pre-cipitation (15) followed by centrifugation for 30 min at 45,000x g. The washed microsomes were suspended in buffer B [50mM sodium phosphate (pH 7.5) containing 20% glycerol and10 mM ME] and stored at -80°C at a protein concentrationof 20-25 mg/ml.

Abbreviations: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; ME, 2-mercaptoethanol.4To whom reprint requests should be addressed.§The sequence reported in this paper has been deposited in theGenBank data base (accession no. L07843).

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Solubilization and Purffication of NADPH-P450 Reductase.Microsomal proteins were solubilized with Emulgen 911 andCHAPS to give a final concentration of2% (vol/vol) Emulgen911 and 2 mg ofCHAPS per mg of microsomal protein. Aftercentrifugation for 60 min at 100,000 x g, the clear supernatantwas loaded onto a DE-52 column previously equilibrated withbuffer C [50 mM sodium phosphate (pH 7:8) with 20%glycerol, 1 mM EDTA, 0.2% Emulgen 911, 0.05% CHAPS,10 mM ME, and 1 ,uM FMN]. The bound NADPH-P450reductase was eluted with a linear gradient of KCl (0.0-0.4M), and the enzymatically active fractions were applieddirectly onto a 2',5'-ADP-Sepharose 4B column (16) that hadbeen equilibrated with buffer D [25 mM sodium phosphate(pH 7.8) with 20% glycerol, 0.1 mM EDTA, 2 mM ME, 0.2%Emulgen 911, 0.05% CHAPS, and 1 ,uM FMN]. The affinity-bound reductase was eluted with 2 mM 2'-AMP in buffer D.The reductase-rich fractions were pooled, concentrated, anddialyzed against buffer E [buffer D containing 10 mM sodiumphosphate buffer (pH 7.8)]. Recombinant rat liver NADPH-P450 reductase was purified as described by Porter et al. (17).SDS/PAGE analyses were performed as described by Laem-mli (18). Proteins were detected by staining with Coomassieblue R-250 or with silver nitrate (19). Western blot analyseswere carried out as described (20). Polyclonal antibodieswere prepared in rabbits and further purified by the methoddescribed (21). Protein concentrations were estimated by thebichoninic acid method (22) using bovine serum albumin as a

standard. The presence of carbohydrate in the purified-plantNADPH-P450 reductase was determined using a GlycoTrackkit obtained from Oxford GlycoSystems. FMN and FADcontents were determined according to the method of Faederand Siegel (23).Enzyme Assays. NADPH-cytochrome c reductase activity

was determined spectrophotometrically at 550 nm as de-scribed (16) using a reaction mixture containing 40 ,uMcytochrome c, 50 mM Tris HCl (pH 7.4), 10 mM MgCl2, 150mM KCl, and 2 mM NaN3. Cinnamic acid 4-hydroxylaseactivity was assayed radiochemically by reversed-phaseHPLC using a C18 ,Bondapak column with a Waters 840HPLC system connected to a radiometer Flo-1 radiodetectorby a method modified from that described by Potts et al. (2).Cytochrome P450 content was determined spectrophotomet-rically (24) using an Aminco DW2 spectrophotometer. Ex-pression of P450 17A in E. coli and preparation ofmembranesfor reconstitution of 17a-hydroxylase activities were similarto those described by Barnes et al. (25) and Fisher et al. (26).Amino Acid Sequence Determination. Amino acid sequence

analyses of the tryptic peptides prepared from purified mungbean reductase were carried out according to the method ofAebersold et al. (27) and Matsudaira (28) in the laboratory ofClive Slaughter (University of Texas Southwestern MedicalCenter at Dallas).

Cloning and DNA Sequencing of Mung Bean SeedlingNADPH-P450 Reductase. Total RNA (10 ,.g), isolated frometiolated mung bean seedlings, was used for the first-strandcDNA synthesis by using the antisense degenerate primerMB2 [5'-GCCTCTAGA(A/G)AA(A/G)TC(A/G)TC(T/C)TC(A/G/T)AT-3'], based on the peptide sequenceIEDDF, attached to a 5' Xba I restriction site. The first-strand cDNA was used as a template for PCR with the sense

degenerate primer MB1 [5'-ITIGCIACITA(C/T)GGNGA-NGG-3'; I = inosine, N = G/A/C/T], based on the peptidesequence (L/M)ATYGDG. PCR was carried out with 800pmol of MB1 and 1200 pmol ofMB2 primers, under standardconditions. Amplification was performed by using a DNAthermal cycler (MJ Research, Watertown, MA) with 30cycles (45 sec at 92°C, 1 min at 42°C, and 2 min at 72°C andan extension for 10 min at 72°C). The identity of the 249-bpPCR product (601-849 bp) was confirmed to be the FMN-binding domain of the P450 reductase by comparing the

deduced amino acid sequence with those of other P450reductases. The 3' region of the reductase cDNA (682-2631bp) was cloned by using the 5' gene-specific primer MB3(5'-ATCTGGCTTCAAAAACTCACC-3') and an oligo(dT)adapter primer as described (29). The 5' region of thereductase cDNA (1-772 bp) was cloned following a methodbased on vector ligation-mediated PCR (K.S. and M.C.M.,unpublished method). The prepared first-strand cDNA usingthe 3' gene-specific primer MB4 (5'-CTTCATCCACTT-TACC-3') was ligated to a vector and used as a template forPCR with a vector primer (T7 or T3) and MB4 primer. Theoverlapping cDNA clones were subcloned in pBluescriptKS' and sequenced on both strands by the chain terminationmethod (30). Restriction enzymes and modification enzymeswere purchased from Promega, Boehringer Mannheim, orPerkin-Elmer/Cetus.

RESULTS AND DISCUSSIONPurification of Mung Bean NADPH-P450 Reductase. The

processing of 500 g of4-day-old mung bean seedlings resultedin the isolation of -3.3 g of microsomal protein. This fractionhad an NADPH-cytochrome c reductase activity of about 40nmol of cytochrome c reduced per min per mg ofprotein. Thespectrophotometric measurement of cytochrome P450showed a content of 4-5 pmol of cytochrome P450 per mg ofprotein. The NADPH-cytochrome c reductase activity ofmicrosomes from fresh plant tissue is -20% of that deter-mined using liver microsomes from untreated rats in whichthe cytochrome P450 content is about 600 pmol of P450 permg of protein. Thus, the ratio of microsomal NADPH-P450reductase to total cytochrome P450 content differs markedlywhen comparing plant tissue with animal tissue (2 and 0.08,respectively). Injury of the hypocotyledons by cutting theminto 1-cm fragments, followed by incubation in a moistatmosphere for 10 h, results in a 3- to 5-fold time-dependentincrease in NADPH-cytochrome c reductase activity andspectrally detectable P450.The procedure for purification of NADPH-P450 (cy-

tochrome c) reductase from the microsomal fraction ofmungbean seedlings (V. radiata) follows the method developed forpurification of the comparable enzyme from liver mi-crosomes (16). The addition of detergents (CHAPS andEmulgen 911) to the microsomal membranes solubilized-95% of the reductase activity. Purification by anion-exchange and affinity chromatography resulted in the '1150-fold purification of the plant NADPH-P450 (cytochrome c)reductase with an -26% yield. The purified protein had aspecific activity of 45-50 gmol of cytochrome c reduced permin per mg of protein.

Properties of the Purified NADPH-P450 Reductase.Coomassie brilliant blue staining of an SDS/PAGE sample ofpurified plant NADPH-P450 reductase showed a major pro-tein band at -82 kDa surrounded by two or more additionalsatellite bands with apparent molecular masses of about 80kDa and 84 kDa (Fig. 1A, lane 6). Comparison with theequivalent enzyme purified from rat liver microsomes (lane 3)indicates a higher mobility for the plant reductase than the ratreductase (about 78 kDa). Previous studies have reported anapparent molecular mass of 82 kDa for the NADPH-P450reductase purified from sweet potato (11) and Jerusalemartichoke (13) and 78 kDa for the NADPH-P450 reductase ofC. roseus (12). Treatment of the plant NADPH-P450 reduc-tase with trypsin resulted in a cleavage to a major band atabout 76 kDa and diffuse bands about 45 kDa (lane 7). Theformation of the 76-kDa protein following tryptic digestion ofthe plant NADPH-P450 reductase indicates the loss of a 5- to6-kDa fragment similar to that seen when rat liver NADPH-P450 reductase is digested with trypsin (lane 4) (31).Western blot analyses were performed as shown in Fig. 1 B

and C. Polyclonal antibodies were prepared against purified

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1 2 3 4 5 6 7 8

A

B

kDa

-9 7.4

-6 6.2

-4 5

-97.4

-66.2

-4 5

-97.4

-6 6. 2

-4 5

c

FIG. 1. SDS/PAGE and Western blot analyses of mung beanseedling NADPH-P450 reductase and rat liver NADPH-P450 reduc-tase. (A) SDS/PAGE gel stained with Coomassie brilliant blue. (B)Western blot using an antibody prepared against rat liver NADPH-P450 reductase. (C) Western blot using an antibody prepared againstpure mung bean seedling NADPH-P450 reductase. Lanes 1 and 8,molecular size markers; lane 2, rat liver microsomes (25 ,ug); lane 3,purified rat NADPH-P450 reductase (0.5 ,ug); lane 4, trypsin-treatedpurified rat NADPH-P450 reductase (0.5 Mg); lane 5, mung beanseedling microsomes (35 ,ug); lane 6, purified mung bean seedlingNADPH-P450 reductase (1.0 Mg); lane 7, trypsin-treated purifiedmung bean seedling NADPH-P450 reductase (1.0 jig).

mung bean seedling NADPH-P450 reductase and rat liverNADPH-P450 reductase, and the IgG fractions were purified.The antibody against rat NADPH-P450 reductase showed across-reaction with the rat liver enzyme (Fig. 1B, lanes 2 and3) but did not cross-react with either the purified or mi-crosome-bound plant NADPH-P450 reductase (Fig. 1B, lanes5 and 6). Use of the antibody prepared against the mung beanNADPH-P450 reductase (Fig. 1C) showed a cross-reactivitywith two or more components in the purified preparation ofmung bean NADPH-P450 reductase at about 82 kDa (lane 6)and reaction with a protein of molecular mass of -76 kDa,indicating the presence of a small amount of degraded enzymein the preparation. The specificity of the antibody against themung bean NADPH-P450 reductase is shown by the absenceof cross-reactivity with rat liverNADPH-P450 reductase (Fig.1C, lanes 2 and 3). Western blot analysis of the trypsin-treatedmung bean and rat liver purified reductases confirmed thisspecificity of cross-reactivity. The immunologically reactiveprotein migrating about 45 kDa in the trypsin-treated sampleof plant reductase probably represents a second trypsin cleav-age product of this enzyme. These observations suggest im-portant differences in the structure of the two orthologousproteins and identify the presence of unique epitopes associ-ated with each protein. The antibody against the mung beanNADPH-P450 reductase was also used to determine immu-nocross-reactivity with comparable enzymes in the microso-mal fraction of other plant tissues, including avocado, soy-bean, alfalfa, and onion. All of these plant microsomal frac-tions showed a positive antibody interaction with proteins ofmolecular masses of 80-84 kDa.

Differences in the immunoreactivity of the plant and ratNADPH-P450 reductases prompted us to investigate differ-

ences in the inhibitory effects of the antibodies. Increasingconcentrations of anti-mung bean NADPH-P450 reductaseIgG inhibited the cinnamic acid 4-hydroxylase activity ofmung bean microsomes, whereas no inhibition was observedwhen equivalent amounts of antibody prepared against ratliver NADPH-P450 reductase were added to the reactionmixture. Likewise, measurements of NADPH-cytochromeP450 (cytochrome c) reductase activities, using either thepurified or the microsomal-bound form of the enzymes,showed the expected profiles of inhibition [i.e., the rate ofcytochrome c reduction by the rat liver NADPH-P450 (cy-tochrome c) reductase was inhibited by the antibody againstthis protein but not by the antibody prepared against themung bean NADPH-P450 reductase and vice versa (data notshown)]. Thus the specificity of the two antibodies wasconfirmed by using either criteria of inhibition of enzymaticfunction or reactivity on Western blot analysis.

Analysis for carbohydrate content of the purified mungbean NADPH-P450 reductase as well as the trypsin-treatedenzyme showed positive pink signals when tested by theGlycoTrack analysis method (32). The glycoprotein nature ofthe purified plant reductase may explain the multiple molec-ular mass forms ofthe plant enzyme, compared to the rat liverenzyme, as recently described by Benveniste et al. (33).The procedure for purification of the mung bean NADPH-

P450 reductase included FMN in the buffers used for chro-matographic purification. A 2- to 3-fold enhancement ofNADPH-cytochrome c reductase activity per mg of proteinwas observed in the presence of exogenously added FMN,suggesting the loss of the FMN prosthetic group duringpurification. The visible optical absorbance spectrum of adialyzed sample of purified plant NADPH-P450 reductaseshowed the typical absorbance spectrum offlavoproteins (i.e.,maxima at 380 nm and 455 nm with a shoulder at 482 nm).Calculation of the flavin content from such spectra, using anextinction coefficient of 11 mM-1cm-1 for the change inabsorbance at 455 nm of the oxidized minus the sodiumdithionite reduced forms ofthe enzyme, indicates the presenceof about 25 nmol offlavin per mg ofprotein. Chemical analysisfor FMN and FAD showed that the dialyzed purified mungbean NADPH-P450 reductase contains 0.62 mol of FAD and0.92 mol of FMN per mole of protein.Measurement of the kinetic properties ofthe purified mung

bean NADPH-P450 (cytochrome c) reductase showed ap-parent Km values for NADPH and cytochrome c of 2.8 ,uMand 7.6 ,M, respectively, at 28°C using 100 mM phosphatebuffer (pH 7.7), which is similar to other plant NADPH-P450reductases (11, 13). No cytochrome c reductase activity wasdetected in presence of NADH.

Sequence Analysis. The full-length nucleotide sequence ofthe mRNA for the mung bean NADPH-P450 reductase wasdetermined from three overlapping cDNA clones, whichtogether contain 2631 nucleotides (data not shown). The 5'untranslated region is 174 bp followed by 2070 bp of an openreading frame and 387 bp of 3' untranslated region. The openreading frame initiates at a methionine codon matching theplant initiator codon consensus sequence (34), and regions ofthe deduced amino acid sequences perfectly match all 15sequenced peptides. The open reading frame encodes apolypeptide of 690 amino acid residues with an estimatedmolecular weight of 76,499 which is similar to the molecularweight of 76,962 of rat liver NADPH-P450 reductase (35).

Fig. 2 presents a comparison of the deduced amino acidsequence ofthe mung bean NADPH-P450 reductase with thecorresponding sequences of comparable enzymes from theplant Arabidopsis, rat liver (35), and yeast (Candida tropi-calis) (36). During the preparation of this manuscript, webecame aware of two DNA nucleotide sequences recentlydeposited in GenBank (ATATRlG and ATATR2M) reportingthe results of studies of two NADPH-P450 reductases from

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Arabidopsis thaliana (unpublished results of C. Mignote-Vieux, M. Kazmaier, F. Lacroute, and D. M. Pompon). TheNADPH-P450 reductase sequences of Arabidopsis(ATATRlG and ATATR2M) have 73% and 67% amino acidsequence identity, respectively, with the mung bean seedlingNADPH-P450 reductase described here. Human, rat, rabbit,and porcine NADPH-P450 reductases have 38.9o, 38.8%,38.3%, and 37.8% amino acid sequence identity, respec-tively, with the mung bean enzyme. Yeast NADPH-P450reductase has a 32.8% amino acid sequence identity with themung bean NADPH-P450 reductase.Mammalian and yeast NADPH-P450 reductases are pro-

posed to consist of five functional domains, including anamino-terminal domain that anchors the protein to the mem-brane and binding regions assigned to the interaction ofFAD,FMN, NADPH, and cytochrome P450 (16, 17, 37). Thesesame features prevail when comparing the amino acid se-quences of the mung bean NADPH-P450 reductase withorthologous proteins from trout, cockroach, human, rabbit,pig, and housefly. Of interest are the clusters of acidic amino

acids (A1-3 of Fig. 2), which may play a role in the electro-static interaction of the NADPH-P450 reductase with thehemoprotein (38). The calculated isoelectric point (pI) for themung bean reductase is 4.95. Potential N-glycosylation sitesare proposed to be amino acid residues 275 and 339. Thisglycosylation may account for the differences in molecularmass and presence of isoforms noted by SDS/PAGE (about80-84 kDa) compared to the calculated molecular mass (76.5kDa) from the amino acid sequence.

Reconstitution of Steroid 17a-Hydroxylase Activities. Therecent success in expressing mammalian P450s in E. coli (25,26) provides a convenient source of different P450s that canbe used for studying the interaction of P450s with differentNADPH-P450 flavoprotein reductases. In this study we havetested the ability of the purified mung bean NADPH-P450reductase to reconstitute the 17a-hydroxylase and 17,20-lyase activities of porcine P450 17A. As shown in Fig. 3A,both 17a-hydroxylase and 17,20-lyase activities were de-tected during the metabolism of progesterone (39) when themung bean NADPH-P450 reductase was used. The effect of

TRYPSINI }--A1--i -FMN-

Vigna 1 MASN ... .SDLVRAVESFLGVSLGDSVSDS.LLLIATTSAAVVVGLLV. .FLWKK.SSDRSKEVKPWVPRDLM. 4EEEEEVDVAAGKTKVTIFFGTQTGT 92Arab 1 MTSALYASDLFKQLKSINMG .. .TDSLSDDVVLVIATTSLALVAGFW.. LLWKKTTADRSGELKPLMIPKSLMAKDEDDDLDLGSGKTRVSI FFGTQTGT 95

Yeast 1 .ALDKLDLYVI ITLWAIAAYF ... AKNQFLDQQQDTGFLNTDSGDGNSRDILQALKKNNKNTLLLFGSQTGT 70Rat 1. MGDSHEDTSATMPEAVAEEVS. LFSTTDMVLFSLIVGVLTYWF. I FRKKKEEIPEFSKIQTTAPPVKESSFVEKMKKTG ..RNI IVFYGSQTGT 90

* * * ****

- FMN -I - FMN I I FMN -IVigna 93 AEGFAKALAEEIIKARYEKAVKVVDLDDYADDDLYEEKLKKESLVFFMLATYGDGEPTDNAARFYKWFTEGKDERGIWLQKLTYGVFGLGNRQYEHFNK 192Arab 96 1EGFAKALSEE9IKARYEKAVKVIDLDDYADDD4YEEKLKKETLAFFCVATYGDGEPTDNARFSKWFTEE.NERDIKLQQLAYGVFALGNRQYEHFNK 194Yeast 71 AEDYANKLSRELHSRFGLKTM.VAD... FADYDFENFGDITEDILVFFIVATYGEGEPTDNADEFHTWLTEEADT ... LSTLKYTVFGLGNSTYEFFNA 162Rat 91 AEEFANRLSKDAH.RYGMRGMSA.DPEEYDLADLSSLPEIDK.SLWFCMATYGEGDPTDMAQDFYDWL... .QETDVDLTGVKFAVFGLGNKTYEHFNA 183

** * * * * * * * **** ****** * * * ** *** ** **

I- A2-f I- A3 1Vigna 193 IGKVVDEELAEQGAKRLVAVGLGDDD.QSIEDDFSAWKESLWSELDQLLRDEDDANTVSTPYTAAILEYRWIHDPT ....A.MSTYDNHSTVANGNTEF 286Arab 195 IGIVLDEELCKKGAKRLIEVGLGDDD.QSIEDDFNAWKESLWSELDKLLKDEDD.KSVATPYTAVIPEYRVVTHDPR. FTTQKSMESNVANGNTTI 287Yeast 163 IGRKFDRLLGEKGGDRFAEYGEGDDGTGtLDEDFLAWKDNVFDSLKNDLNFEEKELKYEPNVKLTERD.DLSGNDPDVSLGEPNVKYIKSEGVDLTKGPF 261Rat 184 MGKYVPQRLEQLGAQRI FELGLGDDD.GNLEEDFITWREQFWPAVCEFFGVE .....ATGEESSIRQYELVVHEDMD.VAKVYTGEMGRLKSYENQKPPF 276

* * * * * * *** ** * * *

I- FAD-1- IVigna 287 DIHHPCRVNVAVQKELHKPESDRSCI HLEFDISGTSITYDTGDHVGVYAENCNETVEETGKLLG._.QNLDLFFSLHTDKDDGTSLGGSLLPPFPGPCSLR 384Arab 288 DIHHPCRVDVAVQKELHTHESDRSCI HLEFDISRTGITYETGDHVGVYAENHVEIVEEAGKLLG. .HSLDLVFSIHADKEDGSPLESAVPPPFPGPCTLG 385Yeast 262 DHTHPFLARIVKTKELFTSE.DRHCVHVEFDISESNLKYTTGDHLAIWPSNSDENIKQFAKCFGLEDKLDTVIELKA .......LDSTYSIPFPNPITYG 353Rat 277 DAKNPFLAAVTANRKLN.QGTERHLMHLELDISDSKIRYESGDHVAVYPANDSALVNQIGEILG ..ADLDVIMSLNNLDE. ESNKKHPFPCPTTYR 368

* * * * * **** * *** * * *****

I- FAD-2-iVi gna 385 TALARYADLLNPPRKAALLALATHASEPSD . ERLKFL. .SSPQGKDEYSKWVVGSQRSLVEVMAEFPSAKPPLGV.. . FFAAIAPRLQPRYYSI SSSPRF 478Arab 386 TGLARYADLLNPPRKSALVALAAYATEPSEAEKLKHL. .TSPDGKDEYSQWIVASQRSLLEVMAAFPSAKPPLGV ... FFAAIAPRLQPRYYSISSCQDW 480Yeast 354 AVIRHHLEISGPVSRQFFLSIAGFAPDEETKKSFTRI .... GGDKQEFASKVTRRKFNIADALLFASNNRPWSDVPFEFLIENVQHLTPRYYSISSSSLS 449Rat 369 TALTYYLDITNPPRTNVLYELAQYASEPSEQEHLHKMASSSGEGKELYLSWVVEARRHILAILQDYPSLRPPID .... HLCELLPRLQARYYSIASSSKV 464

* * * * * * ***** *

I - NADPH-R-RVigna 479 APQRVHVTCALVYGPTPTGRIHKGVCSTWMKNAIPSEKSQDCSS ..... ............API FIRPSNFKLPVDHSIPI IMVGPGTGLAPFRGFLQER 561Arab 481 APSRVHVTSALVYGPTPTGR I HKGVCSTWMKNAVPAEKSHECSG .................API F I RASNFKLPSNPSTPIVMVGPGTGLAPFRGFLQER 563Yeast 450 EKQTINVTAWEAEEEADGRPVTGVVTNLLKNIEIEQNKTGETPMVHYDLNGPRGKFSKFRLPVHVRRSNFKLPKNSTTPVILIGPGTGVAPLRGFVRER 549Rat 465 HPNSVHICAVAVEYEAKSGRVNKGVATSWLRAKEPAGENGGRAL .................VPMFVRKSQFRLPFKSTTPVIMVGPGTGIAPFMGFIQER 547

** ** * * * *** * ***** ** ** **

I NADPH-A IVigna 562 YALKEDGVQLGPALLFfGCRNRQMDFIYEDELKSFVEQ.GSLSELIVAFSREGA.EKEYVQHKMMDKAAHLWSLISQGG.YLYVCGDAKGARDVHRTLH 658Arab 564 MALKEDGEELGSSLLFFGCRNRQMDF6I6YEDELNNFVDQ.GVISELIAFSREGA.QKEYVQHKMEKAAQVWDLIKEEG.YLYVCGDAKGMARDVHRTLH 660Yeast 550 VQQVKNGVNVGKTVLFYGCRNSEQDFLYKQEWSEYASVLGENFEMFNAFSRQODPTKKVYVQDKILENSALVDELLS.SGAI IYVCGDASRMARDVQAAIA 648Rat 548 AWLREQGKEVGETLLYYGCRRSDEDYLYREELARFHKD.GALTQLNVAFSREQA.HKVYVQHLLKRDREHLWKLIHEGGAHIYVCGDARNKAKDVQNTFY 645

* * * *** * * * * **** * *** * * ****** ** **

Vigna 659 SIVQEQENVDSTKAEAIVKKLQMDGRYLRDVW.. 690Arab 661 TIVQEQEGVSSSEAEAIVKKLQTEGRYLRDVW.. 693Yeast 649 KIVAKSRDIHEDKAAELVKSWKVQNRYQEDVW.. 681Rat 646 DIVAEFGPMEHTQAVDYVKKLMTKGRYSLDVWS. 679

** * * ** ***

FIG. 2. Comparison of the deduced amino acid sequence of mung bean (Vigna) NADPH-P450 reductase with corresponding sequences ofArabidopsis (ATATRlG; Arab), Candida tropicalis (yeast), and rat. Sequence alignment was achieved using the program PILEUP developed bythe Genetics Computer Group, permitting the location of common amino acids (marked with an *). The proposed FMN, FAD, andNADPH-binding sites (35) are indicated at the top of the sequences. Clusters of acidic amino acids in the plant reductases are designated asAl, A2, and A3. The location of the amino acid sequences determined by sequencing of tryptic peptides for the design of oligonucleotides areunderlined. The proposed trypsin-sensitive site for cleavage of the hydrophobic membrane insertion sequence is identified by an arrow.

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Proc. Natl. Acad. Sci. USA 90 (1993)

5 0 1 2 5 3

17-OH P4

4 -.

A

AD

CD AL.~

B/ ,

~~~~~~~~0.5nmol P450/mi

4.0 nmol MB Fp/ml

0 5 10 15 20 25 30

Time, min

B 42 0.8o3-

A,0.6

co

cinP45 17 n uiidra ie,rmn baM)NDH0 17ts 00om llse

0- 40

Fp/P450eenMBfor 0m

Foti nin. 5 nTritu ofI( p 7.7),hydroxyand 10m Ccine P450y17A aondentrationsfpurifiedr mung bean (ADPmB PH5

0

Fi fato

tI R c n trisutH o pH r7.5), e10 hyd oxy asand15

with varyingAconcetaiosopurifiedrtlvro mung bean(BNADPH-P5

reductase or recombinant rat NADPH-P450 reductase. [3H]Proges-terone (P4) (5 ,M) was added followed by NADPH (1 mM) asdescribed (25). (A) Time course of the reaction when the ratio ofmung bean NADPH-P450 reductase to cytochrome P450 was 8.17-OH P4, 17a-hydroxyprogesterone; AD, androstenedione. (B)Comparison of activities at varying ratios of flavoprotein (Fp) tocytochrome P450. Activities are expressed as turnover numbers(TN) of the P450 and were calculated from the initial rates ofprogesterone metabolism (17a-hydroxylase; 17-OHase) or the ratesof androstenedione formation (17,20-lyase; lyase).

various concentrations of either the rat or mung beanNADPH-P450 reductases on the turnover ofP450 17A for the17a-hydroxylation of progesterone or the 17,20-lyase reac-tion converting 17a-hydroxyprogesterone to androstenedi-one are summarized in Fig. 3B. Of interest is the greatsimilarity between the two types ofNADPH-P450 reductasesin reconstituting 17a-hydroxylase activity. A small differencewas noted, however, when reconstituting the 17,20-lyaseactivity. The mung bean NADPH-P450 reductase appears tobe a less effective donor of electrons for the lyase reaction.To our knowledge, this is the first report on the heterologousreconstitution of a recombinant mammalian cytochromeP450 with a purified plant NADPH-P450 reductase.The results described in this paper show that the NADPH-

P450 reductase ofmung bean has unique epitopes that permitits distinction from its mammalian counterparts. However,the ability to reconstitute enzymatic activity with a mamma-lian P450 shows that there is sufficient structural similarity inthe plant and mammalian NADPH-P450 reductases to permitelectron transfer from NADPH to the P450. Clearly, theretention of a number of sequences, common over a widerange of phylogenetic orthologs, will permit a better under-standing of structure-function relationships.

We are indebted to Cheryl Martin-Wixtrom, Linda Watkins, and

Jeffrey Laidlaw for technical assistance. The success of this study

was greatly facilitated by the determination of the amino acidsequences of tryptic fragments of the purified enzyme as carried outin the laboratory of Dr. C. A. Slaughter of the Howard HughesMedical Institute, University of Texas Southwestern Medical Cen-ter. This work was supported in part by grants from the CoordinatingBoard of Higher Education of the State of Texas (Advanced Tech-nology Program project no. 003658-201) and the National Institutesof Health (NIGMS-16488) and a Sponsored Research Agreementwith Dallas Biomedical, Inc., awarded to R.W.E. and by grants fromthe National Science Foundation (DCB-8810549) and the TexasAdvanced Technology Program (no. 3474) awarded to M.C.M.

1. Fujita, M., Oba, K. & Uritani, I. (1982) Plant Physiol. 70, 573-578.2. Potts, J. R. M., Weklych, R. & Conn, E. E. (1974) J. Biol. Chem.

249, 5019-5026.3. Benveniste, I. & Durst, F. (1974) C.R. Hebd. Seances, Ser. DAcad.

Sci. 278, 1487-1490.4. Hagmann, M. L., Heller, W. & Grisebach, H. (1983) Eur. J.

Biochem. 134, 547-554.5. Rahier, A. & Taton, M. (1986) Biochem. Biophys. Res. Commun.

140, 1064-1072.6. Madyastha, K. M., Meehan, T. D. & Coscia, C. J. (1976) Biochem-

istry 15, 1097-1102.7. lyanagi, T. & Mason, H. S. (1973) Biochemistry 12, 2297-2308.8. Martin, E. M. & Mortan, R. K. (1955) Nature (London) 176,

113-114.9. Frear, D. S., Swanson, H. R. & Tanaka, F. S. (1969) Phytochem-

istry 8, 2157-2169.10. Ishimaru, I. & Yamazaki, I. (1977) J. Biol. Chem. 252, 199-204.11. Fujita, M. & Asahi, T. (1985) Plant Cell Physiol. 26, 397-405.12. Madyastha, K. M. & Coscia, C. J. (1979) J. Biol. Chem. 254,

2419-2427.13. Benveniste, I., Gabriac, B. & Durst, F. (1986) Biochem. J. 235,

365-373.14. Porter, T. D., Beck, T. W. & Kasper, C. B. (1990) Biochemistry 29,

9814-9818.15. Diesperger, H., Muller, C. R. & Sandermann, H. (1974) FEBS Lett.

43, 155-158.16. Yasukochi, Y., Okita, R. T. & Masters, B. S. S. (1980) Arch.

Biochem. Biophys. 202, 491-498.17. Porter, T. D., Wilson, T. E. & Kasper, C. B. (1987) Arch. Biochem.

Biophys. 254, 353-367.18. Laemmli, U. K. (1970) Nature (London) 227, 680-685.19. Morrissey, J. H. (1981) Anal. Biochem. 117, 307-310.20. Towbin, H., Staehelin, T. & Gordan, J. (1979) Proc. Natl. Acad.

Sci. USA 76, 4350-4354.21. McKinney, M. M. & Parkinson, A. (1987) J. Immunol. Methods 96,

271-278.22. Hill, H. D. & Straka, J. G. (1988) Anal. Biochem. 170, 203-208.23. Faeder, E. J. & Siegel, L. M. (1973) Anal. Biochem. 53, 332-336.24. Omura, T. & Sato, R. (1964) J. Biol. Chem. 239, 2370-2378.25. Barnes, H. J., Arlotto, M. P. & Waterman, M. R. (1991) Proc. Natl.

Acad. Sci. USA 88, 5597-5601.26. Fisher, C. W., Caudle, D. L., Wixtrom, C. M., Quattrochi, L. C.,

Tukey, R. H., Waterman, M. R. & Estabrook, R. W. (1992) FASEBJ. 6, 762-764.

27. Aebersold, R. H., Leavitt, J., Saaverda, R. A., Hood, L. E. &Kent, S. B. H. (1987) Proc. Natl. Acad. Sci. USA 84, 6970-6974.

28. Matsudaira, P. (1987) J. Biol. Chem. 262, 10035-10038.29. Frohmann, M. A., Dush, M. K. & Martin, G. R. (1988) Proc. Natl.

Acad. Sci. USA 85, 8998-9002.30. Sanger, F., Nickelson, S. & Coulson, A. R. (1977) Proc. Natl.

Acad. Sci. USA 74, 5463-5467.31. Vermilion, J. L. & Coon, M. J. (1974) Biochem. Biophys. Res.

Commun. 60, 1315-1322.32. Bayer, E. A., Ben-Hur, H. & Wilchek, M. (1990) Methods Enzymol.

184, 415-427.33. Benveniste, I., Lesot, A., Hasenfratz, M.-P., Kochs, G. & Durst,

F. (1991) Biochem. Biophys. Res. Commun. 177, 105-112.34. Lutcke, H. A., Chow, K. C., Mickel, F. S., Moss, K. S., Kern,

H. F. & Sheele, G. A. (1987) EMBO J. 6, 43-48.35. Porter, T. D. & Kasper, C. B. (1985) Proc. Natl. Acad. Sci. USA

82, 973-977.36. Sutter, T. R., Sangard, D. & Loper, J. C. (1990) J. Biol. Chem. 265,

16428-16436.37. Porter, T. D. & Kasper, C. B. (1986) Biochemistry 25, 1682-1687.38. Shen, S. & Strobel, H. W. (1992) Arch. Biochem. Biophys. 294,

83-90.39. Nakajin, S., Shively, J. E., Yuan, P. M. & Hall, P. F. (1981)

Biochemistry 20, 4037-4042.

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