Proc. Natl. Acad. Sci. USA Vol. 90, pp. 2890-2894, April 1993 Plant Biology Purification, characterization, and cDNA cloning of an NADPH-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 of Botany, University of Texas, Austin, TX 78713 Contributed by Ronald W. Estabrook, December 17, 1992

ABSTRACT We report here the isolation and deduced from microsomes of etiolated mung bean seedlings (Vigna amino acid sequence of the flavoprotein, NADPH-cytochrome radiata). The amino acid sequence, deduced from nucleotide P450 (cytochrome c) reductase (EC 1.6.2.4), associated with the sequences of cDNA clones§, shows that the mung bean microsomal fraction of etiolated mung bean seedlings (Vigna NADPH-P450 reductase shares -38% amino acid sequence radiata var. Berken). An 1150-fold purification of the plant identity with similar NADPH-P450 reductases present in reductase was achieved, and SDS/PAGE showed a predomi- mammalian tissues. Of interest is the presence in the mung nant protein band with an apparent molecular mass of -82 kDa. bean NADPH-P450 reductase of amino acid sequences com- The purified plant NADPH-P450 reductase gave a positive mon with those of orthologous mammalian enzymes and reaction as a glycoprotein, exhibited a typical flavoprotein proposed as binding sites for FMN, FAD, and NADPH (14). visible absorbance spectrum, and contained almost equimolar The purified plant NADPH-P450 reductase is able to function quantities of FAD and FMN per mole of enzyme. Specific efficiently for electron transfer in the reconstitution of the antibodies revealed the presence of unique epitopes distinguish- 17a-hydroxylase and 17,20-lyase activities of mammalian ing the plant and mammalian flavoproteins as demonstrated by P450s with membrane fractions from Escherichia coli ex- Western blot analyses and inhibition studies. Peptide fragments pressing mammalian recombinant P450 17A. from the purified plant NADPH-P450 reductase were se- quenced, and degenerate primers were used in PCR amplifica- MATERIALS AND METHODS tion reactions. Overlapping cDNA clones were sequenced, and FMN, FAD, SDS, 3-[(3-cholamidopropyl)dimethylammonio]- the deduced amino acid sequence of the mung bean NADPH- 1-propanesulfonate (CHAPS), cytochrome c, 2-mercaptoeth- P450 reductase was compared with equivalent enzymes from anol (ME), adenosine 2'-monophosphate (2'-AMP), and soy- mammalian species. Although common flavin and NADPH- bean trypsin inhibitor were purchased from Sigma. Trypsin binding sites are recognizable, there is only ==38% amino acid (L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated) sequence identity. Surprisingly, the purified mung bean was obtained from Worthington. Adenosine 2',5'-diphosphate NADPH-P450 reductase can substitute for purified rat NADPH- (2',5'-ADP)-Sepharose 4B was obtained from Pharmacia. P450 reductase in the reconstitution of the mammalian P450- Polyclar AT (polyvinylpyrrolidone) was a gift from GAF catalyzed 17a-hydroxylation of pregnenolone or progesterone. Chemicals (Wayne, NJ). DE-52 anion-exchange resin and NADPH were purchased from Whatman and Boehringer Higher plants should be a rich source of undiscovered Mannheim, respectively. [3-14C]Cinnamic acid (51 mCi/ cytochrome P450s since plants are recognized to catalyze mmol; 1 Ci = 37 GBq) was obtained from Cen Saclay (Orsay, monooxygenation reactions involved in diverse biosynthetic France). Emulgen 911 was a generous gift from Kao Chemical pathways concerned with the formation of many plant sec- (Tokyo). Ultrogel AcA 34 was purchased from LKB. ondary metabolites [e.g., phytoalexins (1), lignins and fla- Etiolated mung bean seedlings (V. radiata var. Berken) vonoids (2-4), sterols (5), and alkaloids (6), to name but a were purchased locally from Calco (Dallas). The seedlings few]. Microsomal-bound P450 reactions are dependent on the were grown in the dark at 28-30°C and harvested after 4 days. transfer of electrons from NADPH by a common flavopro- All subsequent operations were carried out at 4°C. P450 reductase 1.6.2.4). A Preparation of Microsomes. Fresh seedlings were homog- tein, NADPH-cytochrome (EC enized with a Polytron using 2 ml of buffer A [100 mM unique characteristic of the microsomal NADPH-P450 re- Tris HCl (pH 7.4) containing 250 mM sucrose, 2.8 mM ME, ductase is the presence of noncovalently bound FAD and 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride] FMN as prosthetic groups (7). supplemented with 2% (wt/vol) insoluble Polyclar AT per g Microsomal NADPH-cytochrome c reductase activities wet weight. The homogenate was filtered through nylon cloth have been reported for a number of plants (8-10). The and centrifuged at 25,000 x g for 20 min. The microsomal enzyme has been purified from microsomes of sweet potato fraction was sedimented by centrifugation at 100,000 x g for (11), Catharanthus roseus (12), and Helianthus tuberosus 60 min or by vesicularization using 50 mM MgCl2 for pre- (13) and shown to function in the hydroxylation of the C-10 cipitation (15) followed by centrifugation for 30 min at 45,000 methyl group of geraniol (12) and the p-hydroxylation of x g. The washed microsomes were suspended in buffer B [50 cinnamic acid to form p-coumaric acid (13). The present study was undertaken to compare the enzymes mM sodium phosphate (pH 7.5) containing 20% glycerol and from plants with equivalent enzymes from mammalian and 10 mM ME] and stored at -80°C at a protein concentration bacterial sources. Here we describe the purification and of 20-25 mg/ml. characterization of an NADPH-P450 reductase prepared Abbreviations: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]- 1-propanesulfonate; ME, 2-mercaptoethanol. The publication costs of this article were defrayed in part by page charge 4To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" §The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. L07843).

2890 Downloaded by guest on September 25, 2021 Plant Biology: Shet et al. Proc. Natl. Acad. Sci. USA 90 (1993) 2891 Solubilization and Purffication of NADPH-P450 Reductase. deduced amino acid sequence with those of other P450 Microsomal proteins were solubilized with Emulgen 911 and reductases. The 3' region of the reductase cDNA (682-2631 CHAPS to give a final concentration of2% (vol/vol) Emulgen bp) was cloned by using the 5' gene-specific primer MB3 911 and 2 mg of CHAPS per mg of microsomal protein. After (5'-ATCTGGCTTCAAAAACTCACC-3') and an oligo(dT) centrifugation for 60 min at 100,000 x g, the clear supernatant adapter primer as described (29). The 5' region of the was loaded onto a DE-52 column previously equilibrated with reductase cDNA (1-772 bp) was cloned following a method buffer C [50 mM sodium phosphate (pH 7:8) with 20% based on vector ligation-mediated PCR (K.S. and M.C.M., glycerol, 1 mM EDTA, 0.2% Emulgen 911, 0.05% CHAPS, unpublished method). The prepared first-strand cDNA using 10 mM ME, and 1 ,uM FMN]. The bound NADPH-P450 the 3' gene-specific primer MB4 (5'-CTTCATCCACTT- reductase was eluted with a linear gradient of KCl (0.0-0.4 TACC-3') was ligated to a vector and used as a template for M), and the enzymatically active fractions were applied PCR with a vector primer (T7 or T3) and MB4 primer. The directly onto a 2',5'-ADP-Sepharose 4B column (16) that had overlapping cDNA clones were subcloned in pBluescript been equilibrated with buffer D [25 mM sodium phosphate KS' and sequenced on both strands by the chain termination (pH 7.8) with 20% glycerol, 0.1 mM EDTA, 2 mM ME, 0.2% method (30). Restriction enzymes and modification enzymes Emulgen 911, 0.05% CHAPS, and 1 ,uM FMN]. The affinity- were purchased from Promega, Boehringer Mannheim, or bound reductase was eluted with 2 mM 2'-AMP in buffer D. Perkin-Elmer/Cetus. The reductase-rich fractions were pooled, concentrated, and dialyzed against buffer E [buffer D containing 10 mM sodium RESULTS AND DISCUSSION phosphate buffer (pH 7.8)]. Recombinant rat liver NADPH- Purification of Mung Bean NADPH-P450 Reductase. The P450 reductase was purified as described by Porter et al. (17). processing of500 g of4-day-old mung bean seedlings resulted SDS/PAGE analyses were performed as described by Laem- in the isolation of -3.3 g ofmicrosomal protein. This fraction mli (18). Proteins were detected by staining with Coomassie had an NADPH-cytochrome c reductase activity of about 40 blue R-250 or with silver nitrate (19). Western blot analyses nmol ofcytochrome c reduced per min per mg ofprotein. The were carried out as described (20). Polyclonal antibodies spectrophotometric measurement of cytochrome P450 were prepared in rabbits and further purified by the method showed a content of 4-5 pmol of cytochrome P450 per mg of described (21). Protein concentrations were estimated by the protein. The NADPH-cytochrome c reductase activity of bichoninic acid method (22) using bovine serum albumin as a microsomes from fresh plant tissue is -20% of that deter- standard. The presence of carbohydrate in the purified-plant mined using liver microsomes from untreated rats in which NADPH-P450 reductase was determined using a GlycoTrack the cytochrome P450 content is about 600 pmol of P450 per kit obtained from Oxford GlycoSystems. FMN and FAD mg of protein. Thus, the ratio of microsomal NADPH-P450 contents were determined according to the method of Faeder reductase to total cytochrome P450 content differs markedly and Siegel (23). when comparing plant tissue with animal tissue (2 and 0.08, Enzyme Assays. NADPH-cytochrome c reductase activity respectively). Injury of the hypocotyledons by cutting them was determined spectrophotometrically at 550 nm as de- into 1-cm fragments, followed by incubation in a moist scribed (16) using a reaction mixture containing 40 ,uM atmosphere for 10 h, results in a 3- to 5-fold time-dependent cytochrome c, 50 mM Tris HCl (pH 7.4), 10 mM MgCl2, 150 increase in NADPH-cytochrome c reductase activity and mM KCl, and 2 mM NaN3. Cinnamic acid 4-hydroxylase spectrally detectable P450. activity was assayed radiochemically by reversed-phase The procedure for purification of NADPH-P450 (cy- HPLC using a C18 ,Bondapak column with a Waters 840 tochrome c) reductase from the microsomal fraction of mung HPLC system connected to a radiometer Flo-1 radiodetector bean seedlings (V. radiata) follows the method developed for by a method modified from that described by Potts et al. (2). purification of the comparable enzyme from liver mi- Cytochrome P450 content was determined spectrophotomet- crosomes (16). The addition of detergents (CHAPS and rically (24) using an Aminco DW2 spectrophotometer. Ex- Emulgen 911) to the microsomal membranes solubilized pression of P450 17A in E. coli and preparation of membranes -95% of the reductase activity. Purification by anion- for reconstitution of 17a-hydroxylase activities were similar exchange and affinity chromatography resulted in the '1150- to those described by Barnes et al. (25) and Fisher et al. (26). fold purification of the plant NADPH-P450 (cytochrome c) Amino Acid Sequence Determination. Amino acid sequence reductase with an -26% yield. The purified protein had a analyses of the tryptic peptides prepared from purified mung specific activity of 45-50 gmol of cytochrome c reduced per bean reductase were carried out according to the method of min per mg of protein. Aebersold et al. (27) and Matsudaira (28) in the laboratory of Properties of the Purified NADPH-P450 Reductase. Clive Slaughter (University of Texas Southwestern Medical Coomassie brilliant blue staining of an SDS/PAGE sample of Center at Dallas). purified plant NADPH-P450 reductase showed a major pro- Cloning and DNA Sequencing of Mung Bean Seedling tein band at -82 kDa surrounded by two or more additional NADPH-P450 Reductase. Total RNA (10 ,.g), isolated from satellite bands with apparent molecular masses of about 80 etiolated mung bean seedlings, was used for the first-strand kDa and 84 kDa (Fig. 1A, lane 6). Comparison with the cDNA synthesis by using the antisense degenerate primer equivalent enzyme purified from rat liver microsomes (lane 3) MB2 [5'-GCCTCTAGA(A/G)AA(A/G)TC(A/G)TC(T/ indicates a higher mobility for the plant reductase than the rat C)TC(A/G/T)AT-3'], based on the peptide sequence reductase (about 78 kDa). Previous studies have reported an IEDDF, attached to a 5' Xba I restriction site. The first- apparent molecular mass of 82 kDa for the NADPH-P450 strand cDNA was used as a template for PCR with the sense reductase purified from sweet potato (11) and Jerusalem degenerate primer MB1 [5'-ITIGCIACITA(C/T)GGNGA- artichoke (13) and 78 kDa for the NADPH-P450 reductase of NGG-3'; I = inosine, N = G/A/C/T], based on the peptide C. roseus (12). Treatment of the plant NADPH-P450 reduc- sequence (L/M)ATYGDG. PCR was carried out with 800 tase with trypsin resulted in a cleavage to a major band at pmol of MB1 and 1200 pmol of MB2 primers, under standard about 76 kDa and diffuse bands about 45 kDa (lane 7). The conditions. Amplification was performed by using a DNA formation ofthe 76-kDa protein following tryptic digestion of thermal cycler (MJ Research, Watertown, MA) with 30 the plant NADPH-P450 reductase indicates the loss of a 5- to cycles (45 sec at 92°C, 1 min at 42°C, and 2 min at 72°C and 6-kDa fragment similar to that seen when rat liver NADPH- an extension for 10 min at 72°C). The identity of the 249-bp P450 reductase is digested with trypsin (lane 4) (31). PCR product (601-849 bp) was confirmed to be the FMN- Western blot analyses were performed as shown in Fig. 1 B binding domain of the P450 reductase by comparing the and C. Polyclonal antibodies were prepared against purified Downloaded by guest on September 25, 2021 2892 Plant Biology: Shet et al. Proc. Natl. Acad. Sci. USA 90 (1993)

1 2 3 4 5 6 7 8 kDa ences in the inhibitory effects of the antibodies. Increasing A concentrations of anti-mung bean NADPH-P450 reductase IgG inhibited the cinnamic acid 4-hydroxylase activity of -9 7.4 mung bean microsomes, whereas no inhibition was observed when equivalent amounts of antibody prepared against rat -6 6.2 liver NADPH-P450 reductase were added to the reaction mixture. Likewise, measurements of NADPH-cytochrome P450 (cytochrome c) reductase activities, using either the -4 5 purified or the microsomal-bound form of the enzymes, showed the expected profiles of inhibition [i.e., the rate of cytochrome c reduction by the rat liver NADPH-P450 (cy- tochrome c) reductase was inhibited by the antibody against this protein but not by the antibody prepared against the B -97.4 mung bean NADPH-P450 reductase and vice versa (data not shown)]. Thus the specificity of the two antibodies was -66.2 confirmed by using either criteria of inhibition of enzymatic -4 5 function or reactivity on Western blot analysis. Analysis for carbohydrate content of the purified mung bean NADPH-P450 reductase as well as the trypsin-treated c enzyme showed positive pink signals when tested by the -97.4 GlycoTrack analysis method (32). The glycoprotein nature of -6 6. 2 the purified plant reductase may explain the multiple molec- ular mass forms ofthe plant enzyme, compared to the rat liver -4 5 enzyme, 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- FIG. 1. SDS/PAGE and Western blot analyses of mung bean seedling NADPH-P450 reductase and rat liver NADPH-P450 reduc- matographic purification. A 2- to 3-fold enhancement of tase. (A) SDS/PAGE gel stained with Coomassie brilliant blue. (B) NADPH-cytochrome c reductase activity per mg of protein Western blot using an antibody prepared against rat liver NADPH- was observed in the presence of exogenously added FMN, P450 reductase. (C) Western blot using an antibody prepared against suggesting the loss of the FMN prosthetic group during pure mung bean seedling NADPH-P450 reductase. Lanes 1 and 8, purification. The visible optical absorbance spectrum of a molecular size markers; lane 2, rat liver microsomes (25 ,ug); lane 3, dialyzed sample of purified plant NADPH-P450 reductase purified rat NADPH-P450 reductase (0.5 ,ug); lane 4, trypsin-treated showed the typical absorbance spectrum offlavoproteins (i.e., purified rat NADPH-P450 reductase (0.5 Mg); lane 5, mung bean maxima at 380 nm and 455 nm with a shoulder at 482 nm). seedling microsomes (35 ,ug); lane 6, purified mung bean seedling Calculation of the flavin content from such spectra, using an NADPH-P450 reductase (1.0 Mg); lane 7, trypsin-treated purified extinction coefficient of 11 mM-1cm-1 for the change in mung bean seedling NADPH-P450 reductase (1.0 jig). absorbance at 455 nm of the oxidized minus the sodium mung bean seedling NADPH-P450 reductase and rat liver dithionite reduced forms ofthe enzyme, indicates the presence NADPH-P450 reductase, and the IgG fractions were purified. ofabout 25 nmol offlavin per mg ofprotein. Chemical analysis The antibody against rat NADPH-P450 reductase showed a for FMN and FAD showed that the dialyzed purified mung cross-reaction with the rat liver enzyme (Fig. 1B, lanes 2 and bean NADPH-P450 reductase contains 0.62 mol of FAD and 3) but did not cross-react with either the purified or mi- 0.92 mol of FMN per mole of protein. crosome-bound NADPH-P450 reductase lanes Measurement ofthe kinetic properties ofthe purified mung plant (Fig. 1B, NADPH-P450 5 and Use of the the mung bean bean (cytochrome c) reductase showed ap- 6). antibody prepared against parent Km values for NADPH and cytochrome c of 2.8 ,uM NADPH-P450 reductase (Fig. 1C) showed a cross-reactivity and 7.6 ,M, respectively, at 28°C using 100 mM phosphate with two or more components in the purified preparation of buffer (pH 7.7), which is similar to other plant NADPH-P450 mung bean NADPH-P450 reductase at about 82 kDa (lane 6) reductases (11, 13). No cytochrome c reductase activity was and reaction with a protein of molecular mass of -76 kDa, detected in presence of NADH. indicating the presence of a small amount ofdegraded enzyme Sequence Analysis. The full-length nucleotide sequence of in the preparation. The specificity of the antibody against the the mRNA for the mung bean NADPH-P450 reductase was mung bean NADPH-P450 reductase is shown by the absence determined from three overlapping cDNA clones, which ofcross-reactivity with rat liver NADPH-P450 reductase (Fig. together contain 2631 nucleotides (data not shown). The 5' 1C, lanes 2 and 3). Western blot analysis ofthe trypsin-treated untranslated region is 174 bp followed by 2070 bp of an open mung bean and rat liver purified reductases confirmed this reading frame and 387 bp of 3' untranslated region. The open specificity of cross-reactivity. The immunologically reactive reading frame initiates at a methionine codon matching the protein migrating about 45 kDa in the trypsin-treated sample plant initiator codon consensus sequence (34), and regions of ofplant reductase probably represents a second trypsin cleav- the deduced amino acid sequences perfectly match all 15 age product of this enzyme. These observations suggest im- sequenced peptides. The open reading frame encodes a portant differences in the structure of the two orthologous polypeptide of 690 amino acid residues with an estimated proteins and identify the presence of unique epitopes associ- molecular weight of 76,499 which is similar to the molecular ated with each protein. The antibody against the mung bean weight of 76,962 of rat liver NADPH-P450 reductase (35). NADPH-P450 reductase was also used to determine immu- Fig. 2 presents a comparison of the deduced amino acid nocross-reactivity with comparable enzymes in the microso- sequence ofthe mung bean NADPH-P450 reductase with the mal fraction of other plant tissues, including avocado, soy- corresponding sequences of comparable enzymes from the bean, alfalfa, and onion. All of these plant microsomal frac- plant Arabidopsis, rat liver (35), and yeast (Candida tropi- tions showed a positive antibody interaction with proteins of calis) (36). During the preparation of this manuscript, we molecular masses of 80-84 kDa. became aware of two DNA nucleotide sequences recently Differences in the immunoreactivity of the plant and rat deposited in GenBank (ATATRlG and ATATR2M) reporting NADPH-P450 reductases prompted us to investigate differ- the results of studies of two NADPH-P450 reductases from Downloaded by guest on September 25, 2021 Plant Biology: Shet et al. Proc. Natl. Acad. Sci. USA 90 (1993) 2893 Arabidopsis thaliana (unpublished results of C. Mignote- acids (A1-3 of Fig. 2), which may play a role in the electro- Vieux, M. Kazmaier, F. Lacroute, and D. M. Pompon). The static interaction of the NADPH-P450 reductase with the NADPH-P450 reductase sequences of Arabidopsis hemoprotein (38). The calculated isoelectric point (pI) for the (ATATRlG and ATATR2M) have 73% and 67% amino acid mung bean reductase is 4.95. Potential N-glycosylation sites sequence identity, respectively, with the mung bean seedling are proposed to be amino acid residues 275 and 339. This NADPH-P450 reductase described here. Human, rat, rabbit, glycosylation may account for the differences in molecular and porcine NADPH-P450 reductases have 38.9o, 38.8%, mass and presence of isoforms noted by SDS/PAGE (about 38.3%, and 37.8% amino acid sequence identity, respec- 80-84 kDa) compared to the calculated molecular mass (76.5 tively, with the mung bean enzyme. Yeast NADPH-P450 kDa) from the amino acid sequence. reductase has a 32.8% amino acid sequence identity with the Reconstitution of Steroid 17a-Hydroxylase Activities. The mung bean NADPH-P450 reductase. recent success in expressing mammalian P450s in E. coli (25, Mammalian and yeast NADPH-P450 reductases are pro- 26) provides a convenient source of different P450s that can posed to consist of five functional domains, including an be used for studying the interaction of P450s with different amino-terminal domain that anchors the protein to the mem- NADPH-P450 flavoprotein reductases. In this study we have brane and binding regions assigned to the interaction ofFAD, tested the ability of the purified mung bean NADPH-P450 FMN, NADPH, and cytochrome P450 (16, 17, 37). These reductase to reconstitute the 17a-hydroxylase and 17,20- same features prevail when comparing the amino acid se- lyase activities of porcine P450 17A. As shown in Fig. 3A, quences of the mung bean NADPH-P450 reductase with both 17a-hydroxylase and 17,20-lyase activities were de- orthologous proteins from trout, cockroach, human, rabbit, tected during the metabolism of progesterone (39) when the pig, and housefly. Of interest are the clusters of acidic amino mung bean NADPH-P450 reductase was used. The effect of TRYPSIN I }--A1--i -FMN- Vigna 1 MASN ... .SDLVRAVESFLGVSLGDSVSDS.LLLIATTSAAVVVGLLV. .FLWKK.SSDRSKEVKPWVPRDLM. 4EEEEEVDVAAGKTKVTIFFGTQTGT 92 Arab 1 MTSALYASDLFKQLKSINMG .. .TDSLSDDVVLVIATTSLALVAGFW.. LLWKKTTADRSGELKPLMIPKSLMAKDEDDDLDLGSGKTRVSI FFGTQTGT 95 Yeast 1 .ALDKLDLYVI ITLWAIAAYF ... AKNQFLDQQQDTGFLNTDSGDGNSRDILQALKKNNKNTLLLFGSQTGT 70 Rat 1 . MGDSHEDTSATMPEAVAEEVS. LFSTTDMVLFSLIVGVLTYWF. I FRKKKEEIPEFSKIQTTAPPVKESSFVEKMKKTG ..RNI IVFYGSQTGT 90 * * * ****

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

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

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

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

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

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

Vigna 659 SIVQEQENVDSTKAEAIVKKLQMDGRYLRDVW.. 690 Arab 661 TIVQEQEGVSSSEAEAIVKKLQTEGRYLRDVW.. 693 Yeast 649 KIVAKSRDIHEDKAAELVKSWKVQNRYQEDVW.. 681 Rat 646 DIVAEFGPMEHTQAVDYVKKLMTKGRYSLDVWS. 679 ** * * ** *** FIG. 2. Comparison of the deduced amino acid sequence of mung bean (Vigna) NADPH-P450 reductase with corresponding sequences of Arabidopsis (ATATRlG; Arab), Candida tropicalis (yeast), and rat. Sequence alignment was achieved using the program PILEUP developed by the Genetics Computer Group, permitting the location of common amino acids (marked with an *). The proposed FMN, FAD, and NADPH-binding sites (35) are indicated at the top of the sequences. Clusters of acidic amino acids in the plant reductases are designated as Al, A2, and A3. The location of the amino acid sequences determined by sequencing of tryptic peptides for the design of oligonucleotides are underlined. The proposed trypsin-sensitive site for cleavage of the hydrophobic membrane insertion sequence is identified by an arrow. Downloaded by guest on September 25, 2021 2894 Plant Biology: Shet et al. Proc. Natl. Acad. Sci. USA 90 (1993) was greatly facilitated by the determination of the amino acid sequences of tryptic fragments of the purified enzyme as carried out 17-OH P4 in the laboratory of Dr. C. A. Slaughter of the Howard Hughes Medical Institute, University of Texas Southwestern Medical Cen- A ter. This work was supported in part by grants from the Coordinating Board of Higher Education of the State of Texas (Advanced Tech- 5 0 AD 1 2 5 3 nology Program project no. 003658-201) and the National Institutes of Health (NIGMS-16488) and a Sponsored Research Agreement CD AL.~ with Dallas Biomedical, Inc., awarded to R.W.E. and by grants from 4 -. ~~~~~~~~0.5nmol P450/mi the National Science Foundation (DCB-8810549) and the Texas 4.0 nmol MB Fp/ml Advanced Technology Program (no. 3474) awarded to M.C.M. B/ , 0 5 10 15 20 25 30 1. Fujita, M., Oba, K. & Uritani, I. (1982) Plant Physiol. 70, 573-578. Time, min 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. B 42 0.8 Sci. 278, 1487-1490. o3- A, 0.6 4. Hagmann, M. L., Heller, W. & Grisebach, H. (1983) Eur. J. co 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. 0- 40 8. Martin, E. M. & Mortan, R. K. (1955) Nature (London) 176, 113-114. cinP450 17 n uiidra17ts ie,rmn00ombaM)NDHllse 9. Frear, D. S., Swanson, H. R. & Tanaka, F. S. (1969) Phytochem- istry 8, 2157-2169. Fp/P450eenMBfor 0m 10. Ishimaru, I. & Yamazaki, I. (1977) J. Biol. Chem. 252, 199-204. 11. Fujita, M. & Asahi, T. (1985) Plant Cell Physiol. 26, 397-405. 0 12. Madyastha, K. M. & Coscia, C. J. (1979) J. Biol. Chem. 254, Fi fato 2419-2427. cine P450y17A aondentrationsfpurifiedr mung bean (ADPmB PH5 13. Benveniste, I., Gabriac, B. & Durst, F. (1986) Biochem. J. 235, FotitI nin. 5 R c nnTritutrisutH ofI(o ppH r7.5), e10 7.7),hydroxyhyd oxyand15and0mas 1C 365-373. 14. Porter, T. D., Beck, T. W. & Kasper, C. B. (1990) Biochemistry 29, with varyingAconcetaiosortlvro bean NADPH-P5 purified mung (B 9814-9818. 15. Diesperger, H., Muller, C. R. & Sandermann, H. (1974) FEBS Lett. 43, 155-158. reductase or recombinant rat NADPH-P450 reductase. [3H]Proges- 16. Yasukochi, Y., Okita, R. T. & Masters, B. S. S. (1980) Arch. terone (P4) (5 ,M) was added followed by NADPH (1 mM) as Biochem. Biophys. 202, 491-498. described (25). (A) Time course of the reaction when the ratio of 17. Porter, T. D., Wilson, T. E. & Kasper, C. B. (1987) Arch. Biochem. mung bean NADPH-P450 reductase to cytochrome P450 was 8. Biophys. 254, 353-367. 17-OH P4, 17a-hydroxyprogesterone; AD, androstenedione. (B) 18. Laemmli, U. K. (1970) Nature (London) 227, 680-685. Comparison of activities at varying ratios of flavoprotein (Fp) to 19. Morrissey, J. H. (1981) Anal. Biochem. 117, 307-310. cytochrome P450. Activities are expressed as turnover numbers 20. Towbin, H., Staehelin, T. & Gordan, J. (1979) Proc. Natl. Acad. (TN) of the P450 and were calculated from the initial rates of Sci. USA 76, 4350-4354. progesterone metabolism (17a-hydroxylase; 17-OHase) or the rates 21. McKinney, M. M. & Parkinson, A. (1987) J. Immunol. Methods 96, of androstenedione formation (17,20-lyase; lyase). 271-278. 22. Hill, H. D. & Straka, J. G. (1988) Anal. Biochem. 170, 203-208. various concentrations of either the rat or mung bean 23. Faeder, E. J. & Siegel, L. M. (1973) Anal. Biochem. 53, 332-336. on turnover 24. Omura, T. & Sato, R. (1964) J. Biol. Chem. 239, 2370-2378. NADPH-P450 reductases the ofP450 17A for the 25. Barnes, H. J., Arlotto, M. P. & Waterman, M. R. (1991) Proc. Natl. 17a-hydroxylation of progesterone or the 17,20-lyase reac- Acad. Sci. USA 88, 5597-5601. tion converting 17a-hydroxyprogesterone to androstenedi- 26. Fisher, C. W., Caudle, D. L., Wixtrom, C. M., Quattrochi, L. C., one are summarized in Fig. 3B. Of interest is the great Tukey, R. H., Waterman, M. R. & Estabrook, R. W. (1992) FASEB similarity between the two types ofNADPH-P450 reductases J. 6, 762-764. 27. Aebersold, R. H., Leavitt, J., Saaverda, R. A., Hood, L. E. & in reconstituting 17a-hydroxylase activity. A small difference Kent, S. B. H. (1987) Proc. Natl. Acad. Sci. USA 84, 6970-6974. was noted, however, when reconstituting the 17,20-lyase 28. Matsudaira, P. (1987) J. Biol. Chem. 262, 10035-10038. activity. The mung bean NADPH-P450 reductase appears to 29. Frohmann, M. A., Dush, M. K. & Martin, G. R. (1988) Proc. Natl. be a less effective donor of electrons for the lyase reaction. Acad. Sci. USA 85, 8998-9002. To our knowledge, this is the first report on the heterologous 30. Sanger, F., Nickelson, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. reconstitution of a recombinant mammalian cytochrome 31. Vermilion, J. L. & Coon, M. J. (1974) Biochem. Biophys. Res. P450 with a purified plant NADPH-P450 reductase. Commun. 60, 1315-1322. The results described in this paper show that the NADPH- 32. Bayer, E. A., Ben-Hur, H. & Wilchek, M. (1990) Methods Enzymol. P450 reductase of mung bean has unique epitopes that permit 184, 415-427. its distinction from its mammalian counterparts. However, 33. Benveniste, I., Lesot, A., Hasenfratz, M.-P., Kochs, G. & Durst, F. (1991) Biochem. Biophys. Res. Commun. 177, 105-112. the ability to reconstitute enzymatic activity with a mamma- 34. Lutcke, H. A., Chow, K. C., Mickel, F. S., Moss, K. S., Kern, lian P450 shows that there is sufficient structural similarity in H. F. & Sheele, G. A. (1987) EMBO J. 6, 43-48. the plant and mammalian NADPH-P450 reductases to permit 35. Porter, T. D. & Kasper, C. B. (1985) Proc. Natl. Acad. Sci. USA electron transfer from NADPH to the P450. Clearly, the 82, 973-977. retention of a number of sequences, common over a wide 36. Sutter, T. R., Sangard, D. & Loper, J. C. (1990) J. Biol. Chem. 265, range of orthologs, will permit a better under- 16428-16436. phylogenetic 37. Porter, T. D. & Kasper, C. B. (1986) Biochemistry 25, 1682-1687. standing of structure-function relationships. 38. Shen, S. & Strobel, H. W. (1992) Arch. Biochem. Biophys. 294, 83-90. We are indebted to Cheryl Martin-Wixtrom, Linda Watkins, and 39. Nakajin, S., Shively, J. E., Yuan, P. M. & Hall, P. F. (1981) Jeffrey Laidlaw for technical assistance. The success of this study Biochemistry 20, 4037-4042. Downloaded by guest on September 25, 2021