Glucoside Dhurrin in Sorghum Bicolor

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Proc. Natl. Acad. Sci. USA Vol. 91, pp. 9740-9744, October 1994 Biochemistry Isolation of the heme-thiolate enzyme cytochrome P-450TYR, which catalyzes the committed step in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench (N-hydroxylation/substrate binding spectra/antibody inhibition/dye column chromatography) OLE SIBBESEN, BIRGIT KOCH, BARBARA ANN HALKIER, AND BIRGER LINDBERG M0LLER* Plant Biochemistry Laboratory, Department of Plant Biology, Royal Veterinary and Agricultural University, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark Communicated by Eric E. Conn, May 27, 1994 (receivedfor review April 1, 1994)

ABSTRACT The cytochrome P-450 enzyme (heme- sine to p-hydroxymandelonitrile (5, 6). Sorghum is therefore thiolate enzyme) that catalyzes the N-hydroxylation of L-tyro- a convenient model plant for studying the biosynthesis of sine to N-hydroxytyrosine, the committed step in the biosyn- cyanogenic glucosides. All steps in the biosynthesis of dhur- thesis of the cyanogenic glucoside dhurrin, has been isolated rin except the last are catalyzed by membrane-bound en- from microsomes prepared from etiolated seedlings ofSorghum zymes (5, 7). The two reactions leading to the formation of bicolor (L.) Moench. The cytochrome P-450 enzyme was N-hydroxytyrosine and p-hydroxymandelonitrile are cata- solubilized with the detergents Renex 690, reduced Triton lyzed by enzymes of the cytochrome P450 type (heme- X-100, and 3-[(3-cholamidopropyl)dimethylammonioJ-1- thiolate enzymes) as demonstrated by their (i) dependence on propanesulfonate and isolated by ion-exchange (DEAE- molecular oxygen and NADPH, (ii) inhibition by carbon Sepharose) and dye (Cibacron blue and reactive red 120) monoxide and reversal of the inhibition by 450-nm light, (iii) column chromatography. To prevent irreversible aggregation inhibition by other known cytochrome P450 inhibitors, and of the cytochrome P-450 enzyme, the isolation procedure was (iv) functional inhibition by a specific antibody against the designed without any concentration step-i.e., with dilution of NADPH-cytochrome P-450 oxidoreductase (8). the ion-exchange gel with gel filtration material. The isolated Here we report the isolation of the cytochrome P-450 enzyme, which we designate the cytochrome P-450mYR enzyme, enzyme which catalyzes the N-hydroxylation of tyrosine to gives rise to the specific formation of a ype I substrate binding produce N-hydroxytyrosine. The isolated cytochrome P450 spectrum in the presence of L-tyrosine. The microsomal prep- enzyme, which we designate the cytochrome P-45OryR en- aration contains 0.2 nmol of total cytochrome P-450/mg of zyme, is characterized with respect to its substrate binding protein. The cytochrome P-450TR enzyme is estimated to spectrum and N-terminal amino acid sequence. The isolation constitute :20% of the total cytochrome P-450 content of the procedure described here, which utilizes dye columns, is microsomal membranes and about 0.2% of their total protein gentle and generally applicable for isolation of other cy- content. The apparent molecular mass of the cytochrome tochrome P450 enzymes. P-4501yR enzyme is 57 kDa, and the N-terminal amino acid sequence is ATMEVEAAAATVLAAP. A polyclonal antibody raised against the isolated cytochrome P-450TyR enzyme is MATERIALS AND METHODS specific as monitored by Western blot analysis and inhibits the Chemicals. DEAE-Sepharose fast flow, adenosine 2',5'- in vitro conversion of L-tyrosine to p-hydroxymandelonitrile bisphosphate (2',5'-ADP')-agarose, and Sephacryl S-100 catalyzed by the microsomal system. The cytochrome P-45OTyR were purchased from Pharmacia. Cibacron blue 3GA-agarose enzyme exhibits high substrate specificity and acts as an (type 3000-CL) and reactive red 120-agarose (type 3000-CL) N-hydroxylase on a single endogenous substrate. The reported were obtained from Sigma. Reduced Triton X-100 (RTX) was isolation procedure based on dye columns constitutes a gentle from Aldrich, and Renex 690 was from J. Lorentzen, Kvist- isolation method for cytochrome P-450 enzymes and is of gard, Denmark. All other chemicals used were of reagent general use as indicated by its ability to separate cytochrome grade and purchased from Sigma. P-450wR from the cytochrome P-450 enzyme catalyzing the Preparation of Microsomes. Seeds of S. bicolor (L.) C-hydroxylation ofp-hydroxyphenylacetonitrile and from cin- Moench (hybrid S-1000) were obtained from Seedtec Inter- namic acid 4-hydroxylase. national. (Hereford, TX) and germinated in the dark (2). Microsomes were prepared from '3-cm-high etiolated seed- Seedlings of Sorghum bicolor (L.) Moench contain large lings as described (2), with 2 mM dithiothreitol (DTT) in- amounts of the cyanogenic glucoside dhurrin [,B-D- cluded in all buffers. glucopyranosyloxy-(S)-p-hydroxymandelonitrile] (1) de- Enzyme Assays. Quantitative determination of total cy- rived from the parent amino acid L-tyrosine. Dhurrin is tochrome P450 was carried out by difference spectroscopy synthesized de novo in the seedling (2), and only minute using an extinction coefficient, Ae45o.490, of 91 cm-1 mM-' amounts of dhurrin are present in the seed (3). The key (9) for the adduct between reduced cytochrome P450 and intermediates in the biosynthesis of dhurrin are N-hydroxy- carbon monoxide. Cytochrome P-450 substrate binding spec- tyrosine, p-hydroxyphenylacetaldehyde oxime, p-hydroxy- tra (10) were recorded at a saturating substrate concentration. phenylacetonitrile, and p-hydroxymandelonitrile (4, 5). The The saturating concentration of L-tyrosine (1 mM) was ex- pathway has been elucidated through biosynthetic studies perimentally determined by using the cytochrome P450- using microsomes isolated from etiolated sorghum seedlings. enriched fractions from the DEAE-Sepharose eluate. For The microsomes catalyze the in vitro conversion of L-tyro- Abbreviations: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]- The publication costs of this article were defrayed in part by page charge 1-propanesulfonate; DTT, dithiothreitol; RTX-100: reduced Triton payment. This article must therefore be hereby marked "advertisement" X-100. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 9740 Downloaded by guest on September 24, 2021 Biochemistry: Sibbesen et al. Proc. Natl. Acad. Sci. USA 91 (1994) 9741 detection of cinnamic acid 4-hydroxylase, 1 mM cinnamic column chromatography or of denatured enzyme purified by acid was used. The spectra were recorded on SLM Aminco preparative SDS/PAGE. Freund's complete adjuvant was in- (Urbana, IL) DW-2c and DW-2000 spectrophotometers. cluded in the first injection, and Freund's incomplete adjuvant The effect of antibodies raised against the cytochrome was used in subsequent injections. The immunoglobulin frac- P-450TYR enzyme on biosynthetic activity was measured as tion of the antisera was purified by ammonium sulfate precip- the decrease in cyanide production upon incubation of the itation (12). Western blot analyses were carried out by trans- sorghum microsomes with various substrates. Reaction mix- ferring electrophoresed proteins to nitrocellulose membranes tures (150 p1) contained microsomes (33 ug of protein), and incubation with the cytochrome P45GrYR antibody. Alka- substrate (1.5 ,umol), tricine (7.5 ,umol, pH 8.0), NADPH line phosphatase-conjugated swine antibodies raised against (0.33 ,unmol), and antibodies (0-100 ug of protein). The total rabbit IgG (Dakopatts, Glostrup, Denmark) were used for amount of immunoglobulin in the assay was in each sample vislization. adjusted to 100 ug with purified immunoglobulin from a Other Techniques. SDS/PAGE was carried out in high-Tris nonimmunized rabbit. The antibodies were preincubated 8-25% polyacrylamide linear gradient gels (13). N-terminal with the microsomes for 15 min at 30TC before substrate and amino acid sequencing was performed with an Applied NADPH addition and then incubated at 300C for 30 min. Biosystems model 470A sequenator coupled to a model 120A Cyanide was determined by the Konig reaction (2). Protein phenylthiohydantoin after blotting of the SDS/PAGE- concentration was determined by the method of Bradford purified protein onto ProBlott membrane according to the (11). manufacturer (Applied Biosystems). Sulfide was determined Isolation of the Cytochrome P-4SOTYR Enzyme. All buffers in a spectrophotometric assay after conversion into methyl- were degassed thrice by stirring in vacuo before detergent ene blue (14). and DTT were added. Between each degassing, the buffer was flushed with argon. Subsequent procedures were carried out at 4°C. Microsomes (400 mg of protein in 20 ml, obtained RESULTS from =500 g of seedlings) were diluted to 100 ml with 8.6% The isolation of the cytochrome P-450TYR enzyme is depen- (wt/vol) glycerol/2 mM DTT/10 mM KPi, pH 7.9. The dent on initial solubilization of the total cytochrome P450 microsomes were solubilized by slow addition of 100 ml ofthe content of the sorghum microsomes and subsequent fraction- same buffer fortified with 2% (vol/vol) Renex 690 and 0.2% ation by anion-exchange and dye column chromatography. (vol/vol) RTX-100 and constant stirring for 30 min. Solubi- The recovery of total cytochrome P450 proteins is shown in lized cytochrome P-450 proteins were obtained in the super- Fig. 1. The recovery ofthe cytochrome P4S5rYR enzyme was natant after centrifugation for 30 min at 200,000 x g in a not determined, as accurate substrate binding spectra cannot Beckman 70:Ti rotor. The supernatant (190 ml) was applied be recorded in very crude fractions. A number of detergents (flow rate, 100 ml/hr) to a column (5 cm x 5 cm) of and buffers were tested. Among the detergents, the combined DEAE-Sepharose fast flow/S-100 Sepharose (20:80 wet vol- use of Renex 690, CHAPS, and RTX-100 was found to be umes) equilibrated in buffer A (8.6% glycerol/0.2 mM optimal with respect to maximal recovery of solubilized EDTA/2 mM DTT/1% Renex 690/0.1% RTX-100/10 mM cytochrome P-450 protein and avoidance of the irreversible KPi, pH 7.9). After the column was washed with 150 ml of conversion into cytochrome P-420. The cytochrome P-450 buffer A, most of the cytochrome P-450 proteins, including proteins were labile below pH 7 and above pH 9.5. All buffers the cytochrome P-450ryR enzyme, were eluted with buffer B were fortified with 2 mM DTT. The DTT was stored at {8.6% glycerol/5 mM EDTA/2 mM DTT/1% Renex 690/ 0.1% RTX-100/0.2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)/20 mM KPi, pH 7.9} in a total volume of 250 ml. During this procedure, NADPH- cytochrome P-450 oxidoreductase and cytochrome b5 remain .,), bound to the column and may subsequently be eluted and IsI fractionated by using buffer B fortified with a gradient of 0-300 mM KCl. Final isolation of NADPH-cytochrome P450 oxidoreductase proceeds as in ref. 8. 1.6-e . The cytochrome P-450-containing fractions from the DEAE-Sepharose fast flow/S-100 Sepharose column were 68 - combined (-130 ml) and adjusted to 1% CHAPS, stirred for 30 min, and then applied (flow rate, 40 ml/hr) to a column (2.8 68 - cm x 8 cm) of Cibacron blue-agarose equilibrated in buffer C 38-: (8.6% glycerol/5 mM EDTA/2 mM DTT/1% CHAPS/0.05% RTX-100/20 mM KPi, pH 7.9) fortified with 1% Renex 690. The column was subsequently washed with buffer C until the Renex 690 had been washed out as monitored by measuring the absorbance at 280 nm. The cytochrome P-450TYR enzyme was then eluted with a 0-500 mM KC1 linear gradient (200 ml) in buffer C. The combined cytochrome P-450TYR enzyme I'd~~~~~ fractions were diluted 5-fold with buffer C and applied (flow rate, 7 ml/hr) to a column (0.9 cm x 5 cm) of reactive red 120-Agarose equilibrated in buffer C. The column was washed with 25 ml of buffer C and the cytochrome P-450TYR enzyme was eluted with a 0-1 M KCl linear gradient (60 ml) in buffer C. The yield of the cytochrome P450TYR enzyme FIG. 1. Polypeptide profiles of the fractions obtained during was typically 50-100 pg. isolation of the cytochrome P-45OTyR enzyme as monitored by Antibody Preparation and Western Blot Analysi. Polyclonal SDS/PAGE and staining with Coomassie brilliant blue. Identical antibodies were elicited in rabbits by six repeated subcutaneous amounts of total cytochrome P450 protein (7 pmol) were applied to injections (-15 pg ofprotein per rabbit per injection) at 14-day each lane. The total yields of cytochrome P450 obtained in the intervals of the cytochrome P450ryR enzyme isolated by dye various fractions during a typical isolation experiment are indicated. Downloaded by guest on September 24, 2021 9742 Biochemistry: Sibbesen et al. Proc. Natl. Acad. Sci. USA 91 (1994) -800C, since partly decomposed DTT is contaminated with sulfide (14), which depletes the cytochrome P-450 carbon monoxide spectrum. To prolong the stability of the DTT, all buffers were degassed. Dialysis steps decreased the yield of cytochrome P-450 and were therefore avoided. A major problem encountered in the present study in purifying plant cytochrome P-450 proteins was irreversible aggregation of the proteins, which prevented their subsequent separation by column chromatographic techniques. This problem was over-

come by constantly working with very dilute protein solu- I-- tions and by avoiding concentration steps. To avoid a detri- E mentally high protein concentration upon binding of the 0 cytochrome P-450 proteins to the DEAE column, the DEAE- C4)cI_, agarose was diluted with Sephacryl S-100 gel filtration ma- 0 terial. This caused a marked increase in the separation c efficiency of the DEAE column (Fig. 2) and in addition had a profound positive effect on the separation efficiency sub- ,0(Aq sequently achieved on the dye columns. When the DEAE fractionation step was omitted and the solubilized microsomes were directly applied to the dye columns, all cytochrome P-450 proteins were recovered in the void volume. When used after the DEAE fractionation step, the dye columns retained >90%o of the cytochrome P-450 applied as illustrated in Fig. 3 for the Cibacron blue-agarose column. The ability of the dye columns to separate cytochrome P4SOTYR from cinnamic acid 4-hydroxylase (Fig. 3) and from the cytochrome P-450 enzyme catalyzing the C-hydroxylation ofp-hydroxyphenylacetonitrile (eluted between 0.5 and 0 0.5 1.0 1.0 M KCl) (4, 8) demonstrates the general applicability ofthe KCl, M procedure. The purification of the cytochrome P-450TYR enzyme was FIG. 3. Elution profile of the cytochrome P4STyR enzyme from monitored by its Soret absorption at 420 nm (Fig. 4) and by the Cibacron blue-agarose column. The elution of cytochrome P450 substrate binding spectra (Fig. 5). The isolated cytochrome enzymes was monitored at 420 nm (Soret band). CA-4-H, cinnamic acid 4-hydroxylase; Cyt P-450ox, second cytochrome P450 enzyme P-450TYR enzyme has an apparent molecular mass of 57 kDa, in the biosynthetic pathway for cyanogenic glucosides. (Inset) The which is in the range of other cytochrome P-450 enzymes. A relative position of cytochrome P450ryR (e) and cinnamic acid comparison of the protein profile of the microsomal prepa- 4-hydroxylase (o) as monitored by substrate binding spectra in a ration with that of the isolated cytochrome P-450TYR enzyme separate experiment. shows that the cytochrome P-450TYR enzyme constitutes a very minor portion of the protein content of the microsomal Polyclonal antibodies were raised against the isolated membranes (Fig. 1). The absorption spectrum of isolated cytochrome P-4STYR enzyme and shown to be specific by cytochrome P-450TYR (A. at 421, 541, and 574 nm) (Fig. 4) Western blotting (Fig. 6). To obtain a clearly staining band is typical for that of a low-spin cytochrome P-450 enzyme with the antibody, the gel needs to be overloaded with (Amax at 416, 535, and 565 nm), although redshifted 5-9 nm microsomal protein, again illustrating the low abundance of (10). Addition of tyrosine to the isolated cytochrome the cytochrome P-450rYR protein. When added to the sor- P-450TYR enzyme produced a type I binding spectrum (Amax microsomes, 60% inhibition of the metabolism of at 385 nm, knin at 420 nm) at all tyrosine concentrations tested ghum (Fig. 5). tyrosine was observed at the highest antibody concentration 0.4 0.2 Loading Wash EDTA elution 10.7 nanols P450PP4500 21.4 nmoles P-450

C\OO 0.1 a1) COco Cn J 0 C,)cn

0.0 0 10 20 30 40 50 Fraction FIG. 2. Elution profile from the DEAE-Sepharose/S-100 Seph- arose column. The elution of cytochrome P450 enzymes was monitored at 420 nm (Soret band). The strong absorption during column 700 loading and washing was caused by yellow pigments present in the Wavelength, nm extract. Total cytochrome P450 contents as indicated were determined from the cytochrome P450 carbon monoxide difference FIG. 4. Absorption spectrum of the isolated cytochrome spectrum (9). P-45GrYR enzyme. Downloaded by guest on September 24, 2021 Biochemistry: Sibbesen et al. Proc. Natl. Acad. Sci. USA 91 (1994) 9743

0.03 5 mM tyrosine added to 500 l1l sample -:L- -C. 60 _1 0.02' 90 IL: 30 7

.? ;z -.f :1 7- 0.01 .a 7 1, V); a) co AIA -00

-0.01

65 -0.02- -cxr }-I 8il>1

-0.03 I .- I. 350 400 450 500 Wavelength, nm FIG. 5. Substrate binding spectra of the isolated cytochrome P-450TYR enzyme. tested (see Materials and Methods). The inhibitory effect is specific: no inhibitory effect was observed with p-hydroxyphenylacetaldehyde oxime as substrate, and boiled antibodies have no inhibitory effect. When the isolated cytochrome P-45OTYR enzyme was blot- ted onto ProBlott and the excised protein was subjected to Edman degradation, the following N-terminal amino acid sequence (16 residues) was obtained: ATMEVEAAAATV- LAAP. The initial yield in the sequence analysis was about ((Ov 15 pmol with a repetitive yield of 90%. N\Ncstrnh1.-)()I FIG. 6. The specificity ofthe antibody raised against cytochrome DISCUSSION P450TYR as monitored in Western blot analysis using microsomal It has been proposed that plants have evolved additional membranes and isolated cytochrome P-45OTYR. Identical amounts of biosynthetic to defense-related total cytochrome P450 protein (7 pmol) were applied to each lane. pathways produce secondary For reference, the same amounts of microsomal membranes and plant products and that these pathways often will turn out to cytochrome P-4S5yR were stained with Coomassie brilliant blue. involve highly specific cytochrome P-450 enzymes (15, 16). In response, animals have developed less specific cy- C. roseus, but the function of the proteins represented by tochrome P450-based systems to circumvent the harmful these clones remains unknown (27). effects ofthe secondary plant products. Whereas the involve- None of the cytochrome P-450 enzymes discussed above ment ofcytochrome P-450 enzymes in numerous biosynthetic are N-hydroxylases. The cytochrome P-45OyR enzyme is an pathways leading to secondary plant products has been N-hydroxylase. The absorption spectrum of the isolated demonstrated (17, 18), only afew ofthese proteins have been cytochrome PAS4TYR enzyme is a redshifted low-spin spec- isolated. Pterocarpan 6a-hydroxylase from soybean (Glycine trum (Fig. 4). Upon addition of tyrosine, a type I binding max) was the first cytochrome P-450 enzyme isolated from spectrum is obtained, indicative of the formation of a true plants for which a specific enzymatic activity was demon- substrate-enzyme complex (Fig. 5). The spectral change is strated by reconstitution (19). The cytochrome P450 enzyme saturated at tyrosine concentrations below 1 mM, although allene oxide synthase has been isolated (20) and cloned (21) tyrosine is more hydrophilic than typical substrates for from flax (Linum usitatissimum) seed. Cinnamic acid 4-hy- cytochrome P-450 enzymes and thus is expected to have a droxylase, catalyzing the first hydroxylation step in the relatively low binding affinity. No binding spectra were phenylpropanoid pathway, is a ubiquitous plant cytochrome obtained forp-hydroxyphenylacetonitrile, p-hydroxyphenyl- P450 enzyme and cDNA clones encoding this protein have acetaldehyde oxime, or cinnamic acid, substantiating that been identified (22, 23). A cytochrome P-450 protein has been tyrosine is the endogenous substrate for the cytochrome isolated from ripe avocado (Persea americana) fruits (24). P-45OryR enzyme. Previous in vitro studies using the mi- This cytochrome P450 protein is presumed to be involved in crosomal system have demonstrated that the first step in the monoterpenoid hydroxylations (25), although an endogenous pathway is highly specific, with only tyrosine being metab- substrate has not been identified. Geraniol 10-hydroxylase olized (6). In contrast, the cytochrome P-450 enzymes cata- has been purified from cell suspension cultures of Catharan- lyzing N-hydroxylation ofdrugs and xenobiotics are all rather thus roseus (26). Partial cDNA clones for plant cytochrome nonspecific (28-30). Further molecular characterization of P450 proteins have been isolated from a cDNA library from the cytochrome P-450TYR enzyme may thus provide insight Downloaded by guest on September 24, 2021 9744 Biochemistry: Sibbesen et al. Proc. NatL Acad Sci. USA 91 (1994) into the domains of the active site that determine substrate a continuous free supply of sorghum seeds and Dr. lb Svendsen specificity. (Chemistry Department, Carlsberg Laboratory) for determining the The isolation of cytochrome P-450 enzymes from plant N-terminal amino acid sequence. The work was partially supported tissues has been hampered by their presence in small by the Danish Agricultural and Veterinary Research Council, the amounts and by the presence of plant pigments. Even in Danish International Development Agency, the Danish Center of etiolated plant material, large amounts of yellow pigments Plant Biotechnology, the Carlsberg Foundation, the Rockefeller make quantification ofindividual cytochrome P-450 enzymes Foundation, and the Commission of the European Communities on in crude extracts difficult. In the present study, the content Science and Technology for Development. oftotal cytochrome P-450 of sorghum microsomes was found 1. Akazawa, T., Miljanich, P. & Conn, E. E. (1960) Plant Physiol. to be 0.2 nmol/mg of protein, compared with the earlier 35, 535-538. reported value of 0.10 nmol/mg of protein (31) and corre- 2. Halkier, B. A. & Moller, B. L. (1989) Plant Physiol. 90, sponding to 1% of the total protein content of the mi- 1552-1559. crosomes. The low amount present of the cytochrome 3. Erb, N., Zinsmeister, H. D. & Nahrstedt, A. (1981) Planta P-450TYR enzyme is evidenced by the SDS/PAGE profile of Med. 94, 1219-1224. the microsomal preparation (Fig. 1), which reveals a faint but 4. Halkier, B. A., Lykkesfeldt, J. & Moller, B. L. (1991) Proc. distinct Coomassie brilliant blue-stained band comigrating Natl. Acad. Sci. USA 88, 487-491. with that ofthe isolated cytochrome P-45GryR enzyme. Equal 5. McFarlane, I. J., Lees, E. M. & Conn, E. E. (1975) J. Biol. amounts of total cytochrome P-450 protein were applied to Chem. 250, 4708-4713. the different lanes of the SDS/polyacrylamide gels shown in 6. Moller, B. L. & Conn, E. E. (1979) J. Biol. Chem. 254, Figs. 1 and 6. A comparison of the faint staining intensity of 8575-8583. 7. Halkier, B. A., Olsen, C. E. & Moller, B. L. (1989) J. Biol. the band corresponding to the cytochrome P-45GryR enzyme Chem. 264, 19487-19494. in the microsomal preparation and in the DEAE eluate with 8. Halkier, B. A. & M0ller, B. L. (1991) Plant Physiol. 96, 10-17. the much stronger staining intensity obtained with the iso- 9. Omura, T. & Sato, R. (1964) J. Biol. Chem. 239, 2370-2378. lated enzyme (Fig. 1) indicates that cytochrome P-450TYR 10. Jefcoate, C. R. (1978) Methods Enzymol. 27, 258-279. protein constitutes only about one-fifth of the total microso- 11. Bradford, W. (1976) Anal. Biochem. 72, 248-254. mal cytochrome P-450 content, or about 0.2% of the total 12. Harboe, N. & Ingild, A. (1973) A Manual of Quantitative microsomal protein. A similar ratio between cytochrome Immunoelectrophoresis: Methods and Applications (Univer- P-4STYR and the total microsomal cytochrome P-450 content sitetsforlaget, Oslo). is obtained from the Western blot (Fig. 6). It is remarkable 13. Fling, S. P. & Gregerson, D. S. (1986) Anal. Biochem. 155, that this low amount of cytochrome P45GryR protein can 83-88. mediate an accumulation of dhurrin amounting to 30%o of the 14. H0j, P. B. & Moller, B. L. (1987) Anal. Biochem. 164, 307-314. of the of the 15. Hendry, G. (1986) New Phytol. 102, 239-247. dry weight tip sorghum seedling (2). 16. Nebert, D. W. & Gonzales, F. J. (1987) Annu. Rev. Biochem. Compared with other procedures for isolation of cy- 56, 945-993. tochrome P-450 proteins, the isolation procedure developed 17. Durst, F. (1991) in Frontiers in Biotransformation: Microbial for the cytochrome P-450TYR enzyme is novel with respect to and Plant Cytochromes P450: Biochemical Caracteristics, the use of dye columns. It is important to emphasize that Genetic Engineering and Practical Applications, eds. Ruck- selective binding ofthe cytochrome P-450 enzymes to the dye paul, K. & Rein, H. (Taylor & Francis, Philadelphia), Vol. 4, columns is achievable only after the initial fractionation step pp. 191-232. on the DEAE column has been carried out and that by using 18. Donaldson, R. P. & Luster, D. G. (1991) Plant Physiol. 96, only very dilute protein solutions and by avoiding concen- 669-674. tration steps the method circumvents the tendency of the 19. Kochs, G. & Griesebach, H. (1989) Arch. Biochem. Biophys. cytochrome P-450 proteins to aggregate. A different fraction- 273, 543-553. ation procedure based solely on sucrose gradient centrifuga- 20. Song, W.-C. & Brash, A. R. (1991) Science 253, 781-784. 21. Song, W.-C., Funk, C. F. & Brash, A. R. (1993) Proc. Natl. tion steps in the absence of detergents gave biosynthetically Acad. Sci. USA 90, 8519-8523. active fractions enriched in polypeptides of 50-80 kDa (2). 22. Mizutani, M., Ohta, D. & Sato, R. (1993) Plant Cell Physiol. 34, With the wisdom of hindsight, it can now be concluded that 481-488. these fractions represented a partially purified preparation 23. Teutsch, H. G., Hasenfratz, M. P., Lesot, A., Stoltz, C., containing cytochrome P-450 proteins and NADPH- Gamier, J. M., Jeltsch, J. M., Durst, F. & Werckreichhart, D. cytochrome P-450 oxidoreductase. (1993) Proc. Natl. Acad. Sci. USA 90, 4102-4106. High levels ofcyanogenic glucosides are found in a number 24. O'Keefe, D. P. & Leto, K. J. (1989) Plant Physiol. 89, 1141- ofplants ofwhich several are important food crops (32). Most 1149. important in this respect is the crop plant cassava (Manihot 25. Hallahan, D. L., Nugent, J. H. A., Hallahan, B. J., Dawson, G. W., Smiley, D. W., West, J. M. & Wallsgrove, R. (1992) esculenta) (Crantz), which accumulates the two cyanogenic Plant Physiol. 98, 1290-1297. glucosides linamarin and lotaustralin in its edible roots and 26. Meijer, A. H., Dewaal, A. & Verpoorte, R. (1993) J. Chro- leaves. Mechanical injury of the plant tissue causes hydro- matogr. 635, 237-249. lysis of the cyanogenic glucosides and liberation of cyanide. 27. Meijer, A. H., Souer, E., Verpoorte, R. & Hoge, J. H. C. The presence of cyanogenic glucosides thus dictates careful (1993) Plant Mol. Biol. 22, 379-383. processing ofcassava-derived products before consumption. 28. Fleming, C. M., Branch, R. A., Wilkinson, G. R. & Guenger- Our isolation of the cytochrome P-450 enzyme catalyzing the ich, F. P. (1992) Mol. Pharmacol. 41, 975-980. first committed step in the synthesis of cyanogenic gluco- 29. Miura, S., Degawa, M. & Hashimoto, Y. (1991) Biochem. sides, generation of a monospecific antibody toward this Pharmacol. 42, 1655-1659. 30. Clement, B., Jung, F. & Pfunder, H. (1993) Mol. Pharmacol. enzyme, and determination of the N-terminal amino acid 43, 335-342. sequence ofthe enzyme provide a solid platform for attempts 31. Potts, J. R. M. & Conn, E. E. (1974) J. Biol. Chem. 249, to block or optimize cyanogenic glucoside biosynthesis in 5019-5026. important crop plants. 32. Conn, E. E. (1979) in International Review of Biochemistry: Biochemistry ofNutrition, eds. Neuberger, A. & Conn, E. E. We thank Dr. Jim Foster (Seedtec International, Hereford, TX) for (University Park Press, Baltimore), 1A, Vol. 27, pp. 21-43. Downloaded by guest on September 24, 2021