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Glucoside Dhurrin in Sorghum Bicolor

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Glucoside Dhurrin in Sorghum Bicolor 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.
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