Proc. Natl. Acad. Sci. USAVol. 92, pp. 12505-12509, December 1995Plant Biology

Involvement of cytochrome P450 in oxime production inglucosinolate biosynthesis as demonstrated by an in vitromicrosomal enzyme system isolated from jasmonic acid-induced seedlings of Sinapis alba L.LIANGCHENG DU*, JENS LYKKESFELDT*t, CARL ERIK OLSENt, AND BARBARA ANN HALKIER*§*Plant Biochemistry Laboratory, Department of Plant Biology, and tDepartment of Chemistry, Royal Veterinary and Agricultural University, 40 Thorvaldsensvej,DK-1871 Frederiksberg C. Copenhagen, Denmark

Communicated by Eric E. Conn, University of California, Davis, CA, September 13, 1995 (received for review May 15, 1995)

ABSTRACT An in vitro enzyme system for the conversionof amino acid to oxime in the biosynthesis of glucosinolateshas been established by the combined use of an improvedisolation medium and jasmonic acid-induced etiolated seed-lings of Sinapis alba L. An 8-fold induction of de novo biosyn-thesis of the L-tyrosine-derivedp-hydroxybenzylglucosinolatewas obtained in etiolated S. alba seedlings upon treatmentwith jasmonic acid. Formation of inhibitory glucosinolatedegradation products upon tissue homogenization was pre-vented by inactivation of myrosinase by addition of 100 mMascorbic acid to the isolation buffer. The biosyntheticallyactive microsomal enzyme system converted L-tyrosine intop-hydroxyphenylacetaldoxime and the production of oximewas strictly dependent on NADPH. The Km and Vmax values ofthe enzyme system were 346 ,iM and 538 pmol per mg ofprotein per h, respectively. The nature of the enzyme cata-lyzing the conversion of amino acid to oxime in the biosyn-thesis of glucosinolates has been the subject of much specu-lation. In the present paper, we demonstrate the involvementof cytochrome P450 by photoreversible inhibition by carbonmonoxide. The inhibitory effect ofnumerous cytochrome P450inhibitors confirms the involvement of cytochrome P450. Thisprovides experimental documentation of similarity betweenthe enzymes converting amino acids into the correspondingoximes in the biosynthesis of glucosinolates and cyanogenicglycosides.

Glucosinolates, formerly referred to as mustard oil glucosides,are a group of secondary plant products found mainly withinthe family Brassicaceae (1). Their presence in agricultural cropplants, such as oilseed rape (Brassica napus L.) and brassicaforages, lower the market value of these crops due to thepotentially harmful effects of the breakdown products (2).Although traditional crop breeding has significantly reducedthe overall level of glucosinolates in certain cultivars, tissue-specific elimination of glucosinolates from the seeds of rapeplants is desirable and requires new approaches involvingmolecular techniques.

Glucosinolates are derived from amino acids (3). In vivobiosynthetic studies using seedlings or excised tissues havepreviously shown that N-hydroxyamino acids (4), nitro com-pounds (5), aldoximes (6, 7), thiohydroximates (8), and de-sulfoglucosinolates (8, 9) are precursors of glucosinolates.Cyanogenic glycosides are a related group of secondary plantproducts, which likewise have amino acids as precursors andoximes as intermediates (10). This has suggested a commonbiosynthetic pathway of cyanogenic glycosides and glucosino-lates up to the oxime level (11). Elucidation of the biosyntheticpathway of cyanogenic glycosides has been carried out in vitro

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with highly active microsomal preparations isolated frometiolated seedlings of Sorghum bicolor (L.) Moench, whichproduces the tyrosine-derived cyanogenic glucoside dhurrin(12). In the biosynthesis of dhurrin, a single multifunctionalcytochrome P450 enzyme designated P450tyr has been shownto catalyze the conversion of tyrosine to the correspondingoxime (13).

Isolation of a biosynthetically active in vitro enzyme systemfrom a glucosinolate-producing plant has for decades been amajor goal in glucosinolate research. The efforts have beenunsuccessful because of low de novo biosynthetic activities andas discovered recently because of the release of inhibitorydegradation products from endogenous glucosinolates duringtissue homogenization (14). The latter was monitored bypreparing microsomes from sorghum seedlings in the presenceof leaves of Tropaeolum majus L. containing the glucosinolatebenzylglucosinolate. When prepared separately, the sorghummicrosomes were highly active in catalyzing all except the laststep in synthesis of the cyanogenic glucoside dhurrin. Thebiosynthetic activity of the sorghum microsomes was inacti-vated by the T. majus leaves. The inhibitory component wasshown to be benzylisothiocyanate produced by myrosinasesduring homogenization (14).The nature of the enzymes catalyzing the conversion of

amino acids to oximes in the biosynthesis of glucosinolates hasbeen subject to much speculation. Involvement of flavinmonooxygenases (15, 16), peroxidase-type enzymes (17), andcytochrome P450 enzymes (11) has been suggested. Sinapisalba contains the tyrosine-derived p-hydroxybenzylglucosino-late (p-OHBG) as its major glucosinolate. In this paper, wedescribe a procedure for isolation of biosynthetically activemicrosomes from jasmonic acid-induced etiolated seedlings ofS. alba catalyzing the conversion of tyrosine top-hydroxyphe-nylacetaldoxime. Furthermore, we demonstrate that oximeproduction is dependent on cytochrome P450, as evidenced byphotoreversible inhibition by carbon monoxide. This providesexperimental evidence of similarity between the enzymesconverting amino acids into the corresponding oximes in thebiosynthesis of glucosinolates and cyanogenic glycosides.

MATERIALS AND METHODSMaterials. L-[U-14C]Tyrosine (450 mCi/mmol; 1 Ci = 37 GBq)

was purchased from Amersham. Authentic p-hydroxyphenylac-etaldoxime was chemically synthesized from p-hydroxybenzalde-hyde using hydroxylamine hydrochloride in alkaline medium bythe method of Sekiya et al. (18). The 14C-labeled p-hydroxyphe-

Abbreviations: p-OHBG, p-hydroxybenzylglucosinolate; DTT, dithio-threitol.tPresent address: Department of Pharmacology, University of Copen-hagen, The Panum Institute, 3 Blegdamsvej, DK-2200 CopenhagenN, Denmark.§To whom reprint requests should be addressed.

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nylacetaldoxime was produced enzymatically from L-[U-14C]tyrosine as described (19), and its identity was confirmed bycomparison to the authentic standard. (+)-Jasmonic acid waspurchased from Sigma. p-OHBG was a generous gift fromAnders Kjaer.

Seeds of S. alba were obtained from Prodana Seeds A/S,Odense, Denmark. The seeds were sown and germinated onmoisturized vermiculite at 24°C in complete darkness. After 3days, the -4-cm-high seedlings were sprayed with 50 ,Mjasmonic acid in 50% ethanol and harvested 24 h later.

In Vivo Biosynthesis ofp-OHBG. Excised seedlings of S. albawere fed 1.0 ,uCi of L-[U-14C]tyrosine and incubated for 24 hunder continuous light in transparent boxes to maintain highhumidity. After incubation, the plant material was extracted in90% methanol as described (14). The extracts were concen-trated in vacuo and redissolved in water. Quantitative deter-mination of p-OHBG production was carried out using anHPLC system equipped with UV (LKB-2141) and radioactiv-ity (Berthold LB-506 Cl) monitors. Aliquots (50 ,l) of theextracts were applied to the column (Nucleosil 100-10C18; 250x 4.6 mm) and eluted isocratically with 1% solvent B (70%methanol) in solvent A (0.1 M ammonium acetate) at a flowrate of 1.0 ml/min. The UV absorption of the effluent wasrecorded at 254 nm, and the radioactivity was simultaneouslymonitored by the Berthold monitor fed liquid scintillator(Monoflow 2; National Diagnostics) at a rate of 3.0 ml/min.The retention time ofp-OHBG was verified with an authenticsample. The biosynthetic activity was calculated from theradioactivity incorporated into p-OHBG with correction forthe loss of one carbon atom by decarboxylation. All assays werecarried out in duplicate.

Preparation of the Microsomal Enzyme System. Approxi-mately 100 g of jasmonic acid-induced plant material washomogenized with 10 g of polyvinylpolypyrrolidone, 30 g ofacid-washed sea sand, and 200 ml of isolation buffer [250mMTricine/250 mM sucrose/100 mM ascorbic acid/50 mMNaHSO3/2 mM dithiothreitol (DTT)/2 mM EDTA/1 mMphenylmethylsulfonyl fluoride/5 mg of bovine serum albu-min per ml, pH 8.2] in a prechilled mortar with pestle. Theisolation buffer was degassed and argon-flushed three timesbefore use. The homogenate was filtered through two layersof 22-,um nylon cloth and centrifuged for 10 min at 15,000 xg. The supernatant was centrifuged for 30 min at 200,000 xg. The microsomal pellet was washed once in isolation buffer,and the final pellet was resuspended in a Potter-Elvehjemhomogenizer in the same buffer (-4 ml). The microsomalpreparation was dialyzed sequentially under a nitrogenatmosphere against the isolation buffer for 1 h, against 50mM Tricine, pH 7.9/100 mM ascorbic acid/2 mM DTT for1 h, and twice against 50 mM Tricine, pH 7.9/2 mM DTT for1 h each. After dialysis, the microsomal preparation wasadjusted to a final protein concentration of 10 mg/ml. Theisolation procedure was carried out at 4°C.

In Vitro Biosynthesis ofp-Hydroxyphenylacetaldoxime. Bio-synthetic activity of the microsomal preparation was deter-mined as production of oxime upon administration of tyrosineas substrate. A standard reaction mixture contained 0.4 mg ofprotein, 1.0 ,uCi of L-[U-14C]tyrosine (450 mCi/mmol) dilutedwith 0-1.45 mM unlabeled tyrosine, and 2 mM NADPH in atotal vol of 280 ,ul of 50 mM Tricine, pH 7.9/2 mM DTT. Thereaction was initiated with addition of NADPH. After incu-bation at 35°C for 30 min, the reaction was quenched by theaddition of 750 Al of ethyl acetate. The mixture was centrifugedat 15,000 x g for 5 min. The supernatant was carefully removedand the reaction mixture was reextracted once. The combinedextracts were concentrated in vacuo and redissolved in 40 Al ofethyl acetate for TLC analysis or in 100 ul of 50% ethanol forHPLC analysis. For TLC, an aliquot of the ethyl acetate extractwas applied to TLC plates and the intermediates formed wereseparated in ethyl acetate/toluene (1:5) as described (20).

Radioactive bands on TLC plates were visualized by autora-diography. Production of oxime was quantified by extractingwith ethyl acetate the areas corresponding to authentic oximefollowed by liquid scintillation counting (RackBeta, LKB).HPLC separation of tyrosine, (E)- and (Z)-p-hydroxypheny-lacetaldoxime, p-hydroxyphenylacetonitrile, and p-hydroxy-benzaldehyde was accomplished as reported (19), except thatthe reverse-phase Nucleosil 100-10C18 column was isocraticallyeluted with 4% 2-propanol in 25 mM Tricine (pH 7.9) at a flowrate of 2 ml/min. The retention times of the intermediateswere verified with authentic standards.

Photoreversible Carbon Monoxide Inhibition of OximeProduction. Standard reaction mixtures containing saturatingconcentrations of [14C]tyrosine (200 nmol; 5 mCi/mmol) wereincubated in flat-bottomed glass test tubes sealed with siliconesepta. Each reaction vial was flushed for 10 min with a gascomposed of CO/02/N2 (10:10:80; vol/vol) using injectionneedles inserted in the silicone septum. Tyrosine was injected,the samples were flushed for an additional 1 min, and thereaction was initiated by injection of NADPH. The effect of450-nm light on carbon monoxide inhibition was examined asreported in detail elsewhere (21).

Effects of Cytochrome P450 Inhibitors on Oxime Produc-tion. Microsomal reaction mixtures containing 1.0 ,uCi ofL-[U-14C]tyrosine were incubated in the presence of a series ofcytochrome P450 inhibitors (1 mM) as described in detailelsewhere (21). After 30 min of incubation at 35°C, thereaction mixtures were extracted with ethyl acetate and theextract was analyzed by TLC.GC/MS Analysis. p-Hydroxyphenylacetaldoxime for

GC/MS was obtained by incubating microsomal reactionmixtures with saturating concentrations of unlabeled tyrosine,followed by purification on HPLC and TLC. The isolatedp-hydroxyphenylacetaldoxime was dissolved in ethyl acetateand analyzed on a Hewlett-Packard HP5890 series II gaschromatograph directly interfaced to a JEOL JMS AX505Wmass spectrometer. Splitless injection at 180°C was applied.The capillary column used was a Hewlett-Packard HP-1 (25 m;0.2 mm, i.d.; 0.33-gm film thickness). The head pressure was70 kPa and the oven temperature was programmed as follows:40°C for 1 min, 10°C/min to 250°C, 250°C for 5 min. Electronimpact mass spectra (70 eV; 200-250°C) were obtained at arepetition rate of 1 scan per sec.

RESULTS

Seedlings of several glucosinolate-producing plants testedhave typically had a high content of endogenous glucosinolatesbut very low de novo biosynthetic activity (J.L., unpublishedresults). Thus, administration of [14C]tyrosine to untreated,young seedlings of S. alba resulted in incorporation of 4-7%of the administered radioactivity into p-OHBG. Spraying ofthe seedlings with 50 ,uM jasmonic acid resulted in an 8-foldinduction of the incorporation rate of tyrosine into p-OHBG(Fig. 1). The effect ofjasmonic acid on the de novo biosyntheticactivity of glucosinolates was transient, with maximum induc-tion 24-48 h after treatment, followed by a decrease to normallevels on day 3 after treatment (Fig. 1). Similarly, a 6-foldinduction in the in vitro biosynthesis of oxime by S. albamicrosomes was observed 24 h after jasmonic acid treatment(Fig. 1 Inset). The total content of glucosinolates in the inducedseedlings was not affected significantly.During homogenization of plant material from the glucosi-

nolate-producing plant T. majus, glucosinolates are hydrolyzedby myrosinase to degradation products, which have beenshown to inhibit the enzyme activity involved in cyanogenicglucoside biosynthesis in sorghum microsomes (14). Similarinhibitory effects on sorghum activity have been observedusing S. alba plant material (data not shown). Subsequently, wehave used recovery of the biosynthetic activity of sorghum

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microsomes homogenized in the presence of glucosinolate-containing leaves as a means to optimize the conditions forpreparation of biosynthetically active microsomes from planttissue producing glucosinolates. Ascorbic acid has been shownto stimulate and inhibit myrosinase activity at low (1 mM) andhigh (10 mM) concentrations, respectively (22). In the presentstudy, we used a high concentration of ascorbic acid (100 mM)in both isolation and dialysis buffer to minimize glucosinolatehydrolysis. The procedure of sequential dialysis four timesagainst different buffers, of which the first two contained 100mM ascorbic acid, served to remove glucosinolates from themicrosomal preparation, while myrosinase was kept inacti-vated by ascorbic acid. The addition of bovine serum albumin(5 mg/ml) was found to protect the biosynthetic activity.Furthermore, a buffer capacity as high as 250 mM Tricine (pH8.2) was found necessary to maintain pH at 8.2. Under theseconditions, >85% of sorghum microsomal biosynthetic activitywas recovered when sorghum seedlings were homogenized inthe presence of glucosinolate-containing plant tissue at a ratioof 1:1 (wt/wt) (data not shown).

Using the optimized isolation buffer, it was possible to isolatea biosynthetically active microsomal enzyme system from jas-monic acid-induced etiolated S. alba seedlings. Production of'4C-labeled (E)- and (Z)-oxime in S. alba microsomal reactionmixtures from [14C]tyrosine was demonstrated by comigrationwith the two isomers of authenticp-hydroxyphenylacetaldoximein both TLC and HPLC (Fig. 2). The oxime production wasstrictly NADPH dependent. Unambiguous chemical identifica-tion was accomplished by GC/MS (Fig. 3). On GC, the isolatedp-hydroxyphenylacetaldoxime gave two peaks at retention times17.57 and 17.84 corresponding to the (E)- and (Z)-isomers of theoxime (data not shown). The GC/MS profile of the two peaks wasidentical to authentic p-hydroxyphenylacetaldoxime with a mo-lecular ion of 151 and fragment ions at m/z 107 (tropylium ion)and m/z 133 (M+-H20) (Fig. 3). In vivo administration of14C-labeled oxime resulted in the production of 14C-labeledp-OHBG as evidenced by comigration with authenticp-OHBGon TLC (data not shown). This confirms that oximes are inter-mediates in the biosynthetic pathway of glucosinolates.

In the presence of carbon monoxide, production of p-hydroxyphenylacetaldoxime by S. alba microsomes wasstrongly inhibited by carbon monoxide (Table 1). Upon irra-diation of the carbon monoxide-treated reaction mixture by450-nm light, the inhibition was largely reversed, demonstrat-ing that a cytochrome P450 enzyme is involved in the conver-sion of tyrosine top-hydroxyphenylacetaldoxime. The effect ofputative cytochrome P450 inhibitors on oxime production is

3

E

o

2 - 1 !

1l

,i

U -0

T

i

1 2 3 4 5 6 7 8

Time after treatment, days

FIG. 1. Induction of de novo biosynthesis ofp-OHBG in 3-day-oldseedlings of S. alba upon treatment with jasmonic acid. Biosyntheticactivity was measured as incorporation of radioactivity into p-OHBGupon administration of [U-14C]tyrosine to the seedlings. (Inset) TLCdemonstrating the increase in in vitro biosynthesis of oxime 24 h afterjasmonic acid induction. fw, Fresh weight.

co~~~~~~~~~~~~i

| (Z)-OX=u

I iE,~~(E)-OX

TYR_~~~~~~~~~-NADPH+NADPH

0 10 20 30 40

Time, min

FIG. 2. Production ofp-hydroxyphenylacetaldoxime from tyrosineby the S. alba microsomal enzyme system. Standard reaction mixtureswere incubated with 2.2 nmol of [14C]tyrosine for 30 min at 35°C.p-Hydroxyphenylacetaldoxime accumulated in the reaction mixturesas evidenced by Rf values and retention times equivalent to authenticstandards in TLC (Inset) and HPLC, respectively. OX, p-hydroxyphenylacetaldoxime; TYR, tyrosine.

shown in Fig. 4. Enilkonazol and tetcyclasis are the strongestinhibitors, whereas SK&F 525-A has the least effect. Kineticstudies showed that the enzyme system has a Vm. of 538 pmolof oxime per mg of protein per h and a Km of 346 ,M withrespect to tyrosine.

DISCUSSIONThe present work reports the establishment of an in vitroenzyme system for conversion of amino acids to oximes in thebiosynthesis of glucosinolates using S. alba as a model plant.Isolation of the biosynthetically active microsomal enzyme

system was obtained by the combined use of an improvedisolation buffer and jasmonic-acid induced etiolated seedlings.Jasmonic acid is known to induce plant defense genes, includ-ing genes involved in secondary metabolites (23). It has been

Table 1. Photoreversible carbon monoxide inhibition ofproduction of p-hydroxyphenylacetaldoxime by S. alba microsomes

Experimental Oxime production, %conditions Exp. 1 Exp. 2 Mean + SD

02, - light 100 100 NA02, + light 77 99 88 16CO/02, - light 20 16 18 + 3CO/02, + light 54 73 64 + 14

Data represent two individual experiments. Microsomal reactionmixtures containing 200 nmol of [14C]tyrosine were incubated 30 minat 35°C. Reaction mixtures were extracted with ethyl acetate and theproduced oxime was purified by TLC and quantified by liquid scin-tillation counting. Activity of 100% represents -350 pmol of oxime permg of protein per h. 02, normal atmosphere; CO/02, atmospherecomposed of CO/02/N2 (10:10:80; vol/vol); NA, not applicable.

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107100

50

50 100 150 m/z

FIG. 3. Identification of p-hydroxyphenylacetaldoxime by GC/MS analysis. S. alba microsomes were incubated with saturating amounts oftyrosine in standard reaction mixtures. The oxime produced was extracted with ethyl acetate, purified by TLC, and analyzed by GC/MS.

reported that the content of indole glucosinolates, but notaromatic and aliphatic glucosinolates, can be induced byjasmonic acid in several Brassica species (24, 25). We foundthat de novo biosynthesis ofp-OHBG was transiently induced-8-fold upon treatment with jasmonic acid. The total contentofp-OHBG was not affected, suggesting that the pool of freetyrosine in S. alba might be limiting. Formation of the inhib-itory degradation products fromp-OHBG during microsomalpreparation was prevented by an apparent inhibition of my-rosinase by addition of 100 mM ascorbic acid to all buffers,except those used in the last two dialysis steps after removal ofthe substrate. Comparison of the rate of oxime production bythe S. alba microsomes (Vm~, is 538 pmol of oxime per mg perh) to the, biosynthetic synthetic activity of the sorghum micro-somes producing the tyrosine-derived cyanogenic glucosidedhurrin (400 nmol of HCN per mg of protein per h) (20) showsan -750-fold difference in the activity levels. This illustratespart of the experimental difficulties in obtaining biosyntheti-cally active microsomes from glucosinolate-producing plants.

Recently, Wallsgrove and colleagues (15, 16) have demon-strated a NADPH-dependent 14CO2 release from 14C-labeledhomophenylalanine and dihomomethionine in microsomesprepared from young leaves of oilseed rape (15, 16). GC/MSindicated simultaneous production of the oxime derived fromhomophenylalanine but not of the oxime from dihomomethi-onine (15). The decarboxylation reaction was not inhibited byincubation in 100% carbon monoxide. This indicates thatconversion of amino acids to oximes in the biosynthesis ofglucosinolates does not require molecular oxygen. The inabil-ity of 14C02 production to be inhibited by cytochrome P450inhibitors or 100% carbon monoxide was interpreted by theauthors as an indication that the conversion of amino acid tooxime in the biosynthesis of glucosinolates was not cytochromeP450 dependent (16). Moreover, sensitivity of the decarbox-ylation reaction to DTT and copper salts was interpreted bythe authors as an indication of the involvement of flavinmonooxygenases (16). The reported data are hard to reconcileunless it is assumed that the lack of inhibition by 100% carbon

monoxide reflects the very low levels of activity measuredcombined with incomplete removal of molecular oxygen fromthe reaction mixture before incubation or that the conversionof amino acids to oximes in the biosynthesis of glucosinolatesdoes not require molecular oxygen. In the latter case, flavinmonooxygenases cannot be involved. Alternatively, this mightindicate that the observed decarboxylation reaction does notrepresent the conversion of amino acid to oxime in theglucosinolate biosynthetic pathway. The conversion of L-tryptophan to indole acetaldoxime by a membrane-boundperoxidase-type of enzyme has been demonstrated in micro-somal preparations from several plant species (17). It wasproposed that the enzyme might be important in biosynthesisof indoleacetic acid or maybe indole glucosinolates (17). Thelatter seems unlikely considering that the activity was notrestricted to glucosinolate-producing plants. Based on thesimilarity between the biosynthetic pathways of glucosinolatesand cyanogenic glycosides with respect to amino acid precur-sors and oxime intermediates, it has been speculated thatcytochrome P450 enzymes are involved in oxime production inglucosinolate biosynthesis as it is in cyanogenic glycosidebiosynthesis (11).

In the present report, we have conclusively demonstratedthat the conversion of tyrosine to p-hydroxyphenylacetal-doxime by S. alba microsomes involves cytochrome P450. Thisprovides direct experimental evidence of similarity betweenthe enzymes converting amino acids to oximes in the biosyn-thesis of glucosinolates and cyanogenic glycosides. Wallsgroveet al. (26) have suggested that glucosinolate biosynthesis mayhave evolved independently several times and that differenttypes of enzymes catalyze oxime production in taxonomicallyunrelated species. Based on the observation that cytochromeP450 dependency in oxime production in the biosynthesis ofcyanogenic glycosides has been found in both cyanogenicmono- and dicotyledons (27), we find it likely that the resultsobtained with S. alba can be extended to all glucosinolate-producing plants.

ox

'$** --:-::.,;..- -* "w----_ SlTYR1 2 3 4 5 6 7 8 9

FIG. 4. Effect of cytochrome P450 inhibitors on production ofp-hydroxyphenylacetaldoxime from tyrosine. S. alba microsomes were incubatedwith [14C]tyrosine in the presence of different cytochrome P450 inhibitors (1 mM). After incubation, the oxime was extracted with ethyl acetateand analyzed by TLC. Lanes: 1, without NADPH; 2, no inhibitor; 3, SK&F 525-A; 4, enilkonazol; 5, ancymidol; 6, BAS 110; 7, LAB 150 978; 8,tetcyclasis; 9, BAS 111. OX, p-hydroxyphenylacetaldoxime; TYR, tyrosine.

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,0-SO3N

R S-glucose

Glucosinolates

H NH2

R COOHAmino acids

H NHOH

R COOHN-Hydroxyamino acids

Cyanogenic glycosides

FIG. 5. Comparison of biosynthetic pathways for cyanogenic glycosides and glucosinolates with oxime as the branching point.

In biosynthesis of the cyanogenic glucoside dhurrin, P450tyrhas been shown to catalyze two consecutive N-hydroxylationreactions producing N,N-dihydroxytyrosine, which is proposedto spontaneously dehydrate and decarboxylate to the p-

hydroxyphenylacetaldoxime (13). The identification of cyto-chrome P450tyr as a multifunctional cytochrome P450, com-bined with the demonstration of involvement of cytochromeP450 in oxime production in S. alba microsomes, suggests thata multifunctional cytochrome P450 is involved in the con-version of amino acids to oximes in the biosynthetic pathwayof glucosinolates. In a previous paper, we proposed thataci-nitro compounds were the branching point between thetwo biosynthetic pathways (28). This was based on the abilityof the tautomeric nitro compound to be produced andmetabolized by the sorghum microsomal enzyme system, anobservation that is now known to be a side reaction (13). Inlight of the new data, we now propose that oxime is thebranching point for the biosynthetic pathway of glucosino-lates and cyanogenic glycosides (Fig. 5). Demonstration ofthe involvement of cytochrome P450 in the conversion ofamino acids to oximes in the biosynthesis of glucosinolatesprovides important information for isolation of the corre-sponding genes by molecular techniques.

Professor Anders Kjzer (Department of Organic Chemistry, Tech-nical University of Denmark) and Professor Birger Lindberg M0ller(Department of Plant Biology, The Royal Veterinary and AgriculturalUniversity, Copenhagen) are thanked for helpful discussions. Profes-sor Anders Kjaer is thanked for the generous gift of p-OHBG. Thework was partially supported by The Center for Plant Biotechnology.

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Acad. Sci. USA 88, 487-491.

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