Proc. Nati. Acad. Sci. USAVol. 88, pp. 487-491, January 1991Biochemistry

2-Nitro-3-(p-hydroxyphenyl)propionate and aci-1-nitro-2-(p-hydroxyphenyl)ethane, two intermediates in the biosynthesis of thecyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench

(N-hydroxylation/N-hydroxyTosine/10 incorporation/glucosinolates)

BARBARA ANN HALKIER, JENS LYKKESFELDT, 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, October 1, 1990 (receivedfor review September 4, 1990)

ABSTRACT The biosynthetic pathway for the cyanogenicglucoside dhurrin derived from tyrosine has been studied in vitroby using ['80]oxygen and a microsomal enzyme system obtainedfrom etiolated sorghum seedlings. The products formed werepurified by HPLC and TLC, and the incorporation of ['8O]ox-ygen was monitored by mass spectrometry. In the presence ofNADPH and ['8'Odioxygen, L-tyroSine is converted to (E)- and(Z)-p-hydroxyphenylacetaldehyde oxime with qmantitative in-corporation ofan ['0]oxygen atom into the oxime function. Thefirst step in this conversion is the N-hydroxylation of L-tyrosineto N-hydroxytyrosine. Administration of N-hydroxytyrosine asa substrate results in the production of 1-nitro-2-(p-hydroxyphenyl)ethane in addition to (E)- and (Z)-p-hydroxyphe-nylacetaldehyde oxime, with quantitative incorporation of asingle [O81oxygen atom in all three products. These datademonstrate that the conversion of N-hydroxytyrosine to p-hy-droxyphenylacetaldehyde oxime involves additional N-hydrox-ylation and N-oxidation reactions giving rise to the formation of2-nitro-3-(p-hydroxyphenyl)propionate, which by decarboxyla-tion produces aci-l-nitro-2-(p-hydroxyphenyl)ethane. Bothcompounds are additional intermediates in the pathway. Thetwo [180oxygen atoms introduced by the N-hydroxylations areenzymatically dthable as demonstrated by the specficloss of the oxygen atom introduced by the first N-hydroxylationreaction in the subsequent conversion of aci-1-nitro-2-(p-hydroxyphenyl)ethane to (E)-p-hydroxyphenylactaldehyde ox-ime. A high flux of intermediates through the microsomalenzyme system is obtained with N-hydroxytyrosine as a sub-strate. This renders the conversion of the ad-nitro compoundrate limiting and results in its release from the active site of theenzyme system and accumulation of the tautomeric nitro com-pound.

Sorghum [Sorghum bicolor (L.) Moench] seedlings synthesizethe cyanogenic glucoside dhurrin (fi-D-glucopyranosyloxy-(S)-p-hydroxymandelonitrile) (1). A microsomal enzyme sys-tem obtained from etiolated sorghum seedlings catalyzes the invitro conversion of the parent amino acid tyrosine to thecyanohydrin p-hydroxymandelonitrile (2, 3). Biosyntheticstudies using this experimental system have identified N-hy-droxytyrosine, (E)-p-hydroxyphenylacetaldehyde oxime, (Z)-p-hydroxyphenylacetaldehyde oxime, and p-hydroxyphenyl-acetonitrile as intermediates (2-5). In vivo, a soluble UDP-glucose glucosyltransferase converts the cyanohydrin to thecyanogenic glucoside (6) (Fig. 1). Subsequent studies usingmicrosomal preparations from a number ofother plant specieshave revealed the same biosynthetic pathway (7-11).

Simultaneous measurements of oxygen consumption andtyrosine metabolism using the microsomal enzyme system

isolated from sorghum have shown that three molecules ofoxygen are consumed for each molecule ofp-hydroxymande-lonitrile produced and that the conversion of tyrosine top-hydroxyphenylacetaldehyde oxime proceeds with the con-sumption of two oxygen molecules (12). N-Hydroxytyrosinehas been identified as an intermediate in the latter conversion(3), and its formation consumes one of the two oxygenmolecules. The conversion of N-hydroxytyrosine to p-hy-droxyphenylacetaldehyde oxime represents a two-electronoxidative decarboxylation. The number of possible interme-diates between N-hydroxytyrosine and p-hydroxyphenylace-taldehyde oxime is restricted by the fact that isotope experi-ments using L-[a-2HJtyrosine as substrate have demonstratedquantitative retainment of the a-hydrogen atom of tyrosine inthe p-hydroxyphenylacetaldehyde oxime produced (4). Bio-synthetic studies have demonstrated that the microsomalenzyme system is able to produce and metabolize 1-nitro-2-(p-hydroxyphenyl)ethane and indicate that the nitro com-pound is positioned between N-hydroxytyrosine and p-hy-droxyphenylacetaldehyde oxime (12). However, the amountof 1-nitro-2-(p-hydroxyphenyl)ethane accumulated and me-tabolized is low compared with the amounts observed with thepreviously identified intermediates (2, 3, 5), indicating thepossible involvement of secondary metabolic transformationsnot directly related to the biosynthetic pathway.Hosel et al. (13) have observed the production in vitro of

1-nitro-2-(p-hydroxyphenyl)ethane from tyrosine by microso-mal preparations obtained from osmotically stressed cell sus-pension cultures ofCalifornia poppy (Eschscholtzia CaliforniaCham.). California poppy contains the two tyrosine-derivedcyanogenic glucosides dhurrin and triglochinin. The cell-suspension cultures produce the phenolic glucoside of thenitro compound, whereas they do not produce the cyanogenicglucosides. Conversely, microsomes prepared from 6-day-oldseedlings of California poppy did not produce 1-nitro-2-(p-hydroxyphenyl)ethane (13). The production of the nitro com-pound and its glucoside was concluded to be elicited by theosmotic stress conditions (13).

In this paper, we report the results of incorporation ex-periments with [180dioxygen, which demonstrate that2-nitro-3-(p-hydroxyphenyl)propionate and aci-1-nitro-2-(p-hydroxyphenyl)ethane are obligatory intermediates posi-tioned between N-hydroxytyrosine and p-hydroxyphenyl-acetaldehyde oxime in the biosynthesis of the cyanogenicglucoside dhurrin.

MATERIALS AND METHODSIsotope. [180]Dioxygen (99.2 atom % excess) was pur-

chased from Amersham and stored in a gas burette over asurface of ethylene glycol.

*To whom reprint requests should be addressed.

487

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HO O7H)rHCHH-coOHHOO-Q)HcI4CH-COOH-HOJ)O CHtCH-COOH HOCHf

OH 0' C

L-tyrosine Nhydroxytyrosine 2-nitro-3-(p-hydroxy-phenyl)propionic acid

1-aci-nitro-2-(phydroxy-phenyl)ethane

HO-§ CHiCH2-NOI

1-nitro-2-(p-hydroxy-phenyl)ethane

HO-f CHiCN *- HO-jCHgCsH -

6HHO jC*.C$H

qN-OH

p-hydroxyphenyl-acetonitrile

1rNADPH + OgWKAPG

(Z)-p-hydroxyphenyl-acetaldehyde oxime

(E)-p-hydroxyphenyl-acetaldehyde oxime

in vitro in vivoHO-FCHO + HCN -* HO CH-CN HO- o9CCN

6=H \= 6-gluome

p-hydroxy- hydrogenbenzaldehyde cyanide

p-hydroxy-mandelonitrile

dhurrin

FIG. 1. The biosynthetic pathway for the tyrosine-derived cyanogenic glucoside dhurrin from S. bicolor. The intermediates positionedbetween N-hydroxytyrosine and (E)-p-hydroxyphenylacetaldehyde oxime are identified in this study.

Chemical Syntheses. (R,S)-N-Hydroxytyrosine was syn-thesized by chemical reduction of p-hydroxyphenylpyruvicacid oxime with sodium cyanoborohydride (14). (E)- and(Z)-p-hydroxyphenylacetaldehyde oxime were synthesizedin an =1:1 ratio by oxidative decarboxylation of N-hydroxy-tyrosine in 1 M NH3 (15). Each of the isomers was isolatedfrom the reaction mixture by reverse-phase HPLC (4). 1-Ni-tro-2-(p-hydroxyphenyl)ethane was synthesized by sodiumborohydride reduction of 1-nitro-2-(p-hydroxyphenyl)etheneobtained by condensation of p-hydroxybenzaldehyde andnitromethane (16).

Preparation of the Microsomal Enzyme System. The mi-crosomal enzyme system was prepared from etiolated seed-lings of S. bicolor (L.) Moench (hybrid Hybridum) (SeedtecInternational, Hereford, TX) as earlier described (1, 3). Whenprepared in the presence of dithiothreitol, the microsomalenzyme system catalyzes the conversion of tyrosine to p-hy-droxymandelonitrile (2, 3). When prepared in the absence ofdithiothreitol and subjected to dialysis for 2 days in theabsence of dithiothreitol, the microsomal enzyme systemselectively catalyzes the conversion of tyrosine to the ste-reoisomeric p-hydroxyphenylacetaldehyde oximes (2, 17).The latter type of preparation was used in the present study.

['80]Oxygen Labeling Experiments. The biosynthetic reac-tions were carried out in septum-covered glass reactionvessels (5 ml) fitted with an inlet for a vacuum line and onefor the administration of ['8O]dioxygen. The pH and type ofbuffer used in the biosynthetic reaction mixtures were variedto optimize the metabolic conversion rate for each specificsubstrate used (3). N-Hydroxytyrosine (1.50 ,umol) was ad-ministered to reaction mixtures containing 200 ,ul of microso-mal enzyme system (2.0 mg of protein) and 30 ,mol ofpotassium phosphate (pH 6.5) in a total volume of 530 ,ul.Tyrosine, 1-nitro-2-(p-hydroxyphenyl)ethane, or p-hydroxy-phenylacetaldehyde oxime (0.55 umol) was administered toreaction mixtures containing 240 ,ul of microsomal enzymesystem (2.4 mg of protein) and 5 ,umol of N-tris(hydroxy-methyl)methylglycine (Tricine) (pH 7.9) in a total volume of530 ,l. After flushing with argon for 5 min and subsequentevacuation (13 mm Hg; 1 mm Hg = 133 Pa), ['80]dioxygen

was admitted from the gas burette until atmospheric pressurewas obtained in the reaction vessels. NADPH (0.90 ,tmol)was injected, and the reaction mixture was incubated 1 hr at30°C. At the end of the incubation period, accumulatedintermediates were separated by direct injection of the reac-tion mixture onto a reverse-phase HPLC column (Fig. 2) (4).The HPLC effluents containing 1-nitro-2-(p-hydroxyphe-nyl)ethane and (E)- and (Z)-p-hydroxyphenylacetaldehydeoximes were collected and lyophilized, and the intermediateswere purified by TLC [Merck F254 aluminum plates; hexane/ether, 1:3 (vol/vol)]. Mass spectra were recorded on a VGMasslab TRIO-2 mass spectrometer.

RESULTSThe mass spectra of 1-nitro-2-(p-hydroxyphenyl)ethane andp-hydroxyphenylacetaldehyde oxime display prominent mo-lecular ions [M]+ at m/z 167 and 151, respectively. Theincorporation of [180]oxygen into the nitro compound and the

£ TYR CHO

C~~~~C-I' I|o (E)OXa0 5 10 15 20 25 30

Retention time (min)

FIG. 2. Separation by reverse-phase HPLC of reference com-pounds representing those involved in dhurrin biosynthesis. Theseparation was carried out using a Nucleosil C18 (10-Am) column andisocratic elution with 2% 2-propanol in 25 mM Hepes (pH 7.9) (4).TYR, tyrosine; CHO, p-hydroxybenzaldehyde; CN, p-hydroxyphe-nylacetonitrile; OX, p-hydroxyphenylacetaldehyde oxime; NO2,1-nitro-2-(p-hydroxyphenyl)ethane. Neither CHO nor CN is pro-duced under the experimental conditions used in this study.

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oxime can therefore be quantitatively assessed from the shiftin intensities of the molecular ions [M]+ from m/z 167 to 169and from 151 to 153, respectively. The diagnostic fragmentions [HO-Ph-CH=CH21' at m/z 120 and [HO-Ph-CH2]+ atm/z 107 constitute the base peak in the spectrum of the nitrocompound and the oxime, respectively. The masses of thesefragment ions remained at m/z 120 and 107, regardless ofwhether the isolation of the nitro compound and the oximefrom biosynthetic experiments was carried out in an 1802 or1602 atmosphere. This demonstrates that with the substratesused exchange reactions resulting in incorporation of[18O]oxygen into the aromatic hydroxyl group do not occur.In the biosynthetic incorporation experiments, all relativeintensities are corrected for the 0.2% natural abundance ofthe 180 isotope.

Tyrosine and N-hydroxytyrosine were used as substratesin the biosynthetic experiments together with [18O]dioxygen(Table 1). The [18O]dioxygen enrichment of the atmospherein the reaction vessels was 90.5 ± 0.1 atom % excess asmeasured by gas mass spectrometry. When tyrosine is usedas substrate in the presence of NADPH and 1802, HPLCanalyses show the accumulation of the (E)- and (Z)-p-hydroxyphenylacetaldehyde oximes in the reaction mixture,whereas no 1-nitro-2-(p-hydroxyphenyl)ethane is detectable.The mass spectra of the (E)- and (Z)-p-hydroxyphenylace-taldehyde oximes isolated from the reaction mixtures areidentical to those of the authentic compounds except forshifts related to incorporation ofan [180]oxygen atom into theoxime group. For the (E)- and (Z)-p-hydroxyphenylacetalde-hyde oximes, the enrichment based on the intensities of them/z 153 and m/z 151 molecular ions is 89.1% and 88.1%,respectively. These two molecular ions exhibit identicalevaporation profiles. Thus incorporation of [18O]oxygen intothe oxime function takes place without dilution, demonstrat-ing that dioxygen serves as the sole source for this oxygenatom.

Administration of N-hydroxytyrosine to the microsomalenzyme system in the presence of NADPH and [180]dioxy-gen results in the accumulation of 1-nitro-2-(p-hydroxyphe-nyl)ethane and (E)- and (Z)-p-hydroxyphenylacetaldehydeoxime. In some experiments, the amount of the nitro com-pound produced equals that ofthe oxime isomers, whereas inother experiments it is somewhat lower. The fragmentationpattern of the isolated nitro compound is identical to that ofthe chemically synthesized standard, except for differencesattributed to the incorporation of an [180]oxygen atom intothe nitro group. Thus the molecular ion was shifted to [M]+m/z 169 with an enrichment of 88.0% based on the intensitiesof the m/z 169 and m/z 167 ions. These ions also haveidentical evaporation profiles. The incorporation of [180]ox-

Table 1. Incorporation of [18O]oxygen into components producedby the microsomal enzyme system in incubation atmospherescontaining [180]dioxygen

[180]Oxygenenrichment of

isolated intermediate, Dilution ofatom % excess [180]oxygen

Substrate NO2 (E)-OX (Z)-OX NO2 (E)-OX (Z)-OXL-Tyrosine - 89.1 88.1 1.02 1.03N-Hydroxytyrosine 88.0 29.8 26.9 1.03 3.04 3.36N-Hydroxytyrosine 1.22* 1.20*

Dilution of [180]oxygen was calculated as the ratio of the [180]ox-ygen content of the incubation atmosphere to that of the intermedi-ates isolated. The [180]dioxygen enrichment of the incubation at-mosphere was 90.5 atom % excess. NO2, 1-nitro-2-(p-hydroxyphe-nyl)ethane; OX, p-hydroxyphenylacetaldehyde oxime.*Corrected for p-hydroxyphenylacetaldehyde oxime produced dueto chemical decomposition of N-hydroxytyrosine.

ygen without any dilution demonstrates that the conversionof N-hydroxytyrosine to 1-nitro-2-(p-hydroxyphenyl)ethaneproceeds with quantitative incorporation of an oxygen atomderived from dioxygen.As in the experiments with tyrosine as a substrate, the

mass spectra of the (E)- and (Z)-p-hydroxyphenylacetalde-hyde oximes isolated from the reaction mixtures with N-hy-droxytyrosine as substrate are identical to those of theauthentic standards except for the shifts related to incorpo-ration of an [180]oxygen atom into the oxime group. Theenrichment based on the intensities of the m/z 153 and m/z151 molecular ions is 29.8% and 26.9% for the E and Zisomers, respectively, corresponding to dilution factors of3.04 and 3.36. The dilution reflects the previously reportedchemical decomposition of N-hydroxytyrosine into p-hy-droxyphenylacetaldehyde oxime, which occurs along withthe enzymatic conversion (15). The amount of (E)- and(Z)-p-hydroxyphenylacetaldehyde oximes formed by chem-ical decomposition was determined in control experiments inwhich NADPH was omitted from the incubation mixture andwas found to constitute 60% and 64%, respectively, of theamount of E and Z isomers formed in the biosyntheticexperiments carried out in the presence ofNADPH. We haveearlier reported that chemical decomposition of N-hydroxy-tyrosine produces the E and Z isomers in an -1:1 ratio (4).In the present experiment the ratio determined by the HPLCanalysis was 1.57:1. The microsomal enzyme system initiallyproduces the E isomer, which subsequently is enzymaticallyconverted to the Z isomer (4). Generally, the ratio betweenthe enzymatically produced E and Z isomers varies between2.6 and 3.3 (4). This ratio explains why the calculated dilutionfactor is higher for the Z isomer compared with the E isomer.When the dilution factors are corrected for the contributionof the chemically formed oxime isomers, dilution factors of1.22 and 1.20 are obtained. These dilution factors demon-strate an almost quantitative incorporation of an [180]oxygenatom derived from dioxygen into each of the two oximeisomers when N-hydroxytyrosine is used as substrate andunequivocally establishes that the enzymatic conversion ofN-hydroxytyrosine to p-hydroxyphenylacetaldehyde oximerequires an N-oxidation reaction. It also demonstrates thatthe oxygen atom of the hydroxyamino group of N-hydroxy-tyrosine is lost in the overall conversion of N-hydroxyty-rosine to p-hydroxyphenylacetaldehyde oxime.Except for the chemical decomposition of N-hydroxyty-

rosine into the stereoisomeric p-hydroxyphenylacetaldehydeoximes, the production of 1-nitro-2-(p-hydroxyphe-nyl)ethane and (E)-p-hydroxyphenylacetaldehyde oxime inthe biosynthetic reaction mixtures is strictly dependent onthe presence of NADPH. Incubation of N-hydroxytyrosine,1-nitro-2-(p-hydroxyphenyl)ethane, and p-hydroxyphenyl-acetaldehyde oxime with the microsomal system in an 1802atmosphere in the presence or absence of NADPH andsubsequent reisolation ofthe administered substrate revealedthat exchange reactions leading to the incorporation of[180joxygen in the substrates do not occur. Similarly, chem-ical decomposition of N-hydroxytyrosine to p-hydroxyphe-nylacetaldehyde oxime proceeds without any incorporationof [180]oxygen. In no case did the administration ofthe oximeisomers to the microsomal enzyme system result in theproduction of the nitro compound.

DISCUSSIONIn the present study, we have used incorporation experi-ments with the stable isotope [180]oxygen to conclusivelydemonstrate the obligatory involvement of an N-oxidationreaction in the enzymatic conversion of N-hydroxytyrosineto p-hydroxyphenylacetaldehyde oxime. Taken togetherwith the initial N-hydroxylation reaction of tyrosine to N-hy-

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droxytyrosine (3), these data explain the previously reportedconsumption of two oxygen molecules in the conversion oftyrosine to (E)- and (Z)-p-hydroxyphenylacetaldehyde oxime(12).The interpretation of the previously obtained biosynthetic

data on the nitro compound was ambiguous due to the lowmetabolic conversion rate of 1-nitro-2-(p-hydroxyphenyl)-ethane when compared with the established intermediates.The Vmax value for the nitro compound is 14 nmol per hr permg of protein (12) compared with values of 145, 345, and 400nmol per hr per mg of protein for tyrosine, N-hydroxyty-rosine, and p-hydroxyphenylacetaldehyde oxime, respec-tively (3). Only minute amounts of the nitro compoundaccumulated in biosynthetic reaction mixtures with tyrosineas substrate (12). In the present study, the use ofN-hydroxy-tyrosine as a substrate resulted in the accumulation ofsignificant amounts of the nitro compound. This indicatesthat the initial N-hydroxylation of tyrosine to produce N-hy-droxytyrosine is the overall limiting reaction in the pathway.At the increased flux of intermediates obtained with N-hy-droxytyrosine as substrate, a later step in the conversionpresumably becomes rate limiting as indicated by the accu-mulation of 1-nitro-2-(p-hydroxyphenyl)ethane. When pro-duced from N-hydroxytyrosine in an [18O]dioxygen atmo-sphere, one of the oxygen atoms of the nitro group is labeledwithout any dilution (Table 1). In the same reaction mixtures,an [18O]oxygen atom is almost quantitatively incorporatedinto the oxime produced. The latter result demonstrates thatthe two oxygen atoms introduced by the two successiveN-oxidation reactions are enzymatically distinguishablethroughout the subsequent enzymatic transformations. Thisexcludes 1-nitro-2-(p-hydroxyphenyl)ethane as a freely dif-fusible intermediate in the pathway.Only one biosynthetic route accounts for the accumulation

of the nitro compound in the conversion of N-hydroxyty-rosine to p-hydroxyphenylacetaldehyde oxime and at thesame time is consistent with the previously obtained biosyn-thetic data. This pathway involves the N-oxidation of N-hy-droxytyrosine giving rise to the formation of 2-nitro-3-(p-hydroxyphenyl)propionate. This conversion represents afour-electron oxidation composed of the dioxygen andNADPH-dependent hydroxylation reaction and a two-electron oxidation reaction. If the two-electron oxidationreaction precedes the N-hydroxylation, then the substrate forthe N-hydroxylation reaction is 3-(p-hydroxyphenyl)-2-nitrosopropionate. Due to their chemical lability, it is notpossible to directly test the a-nitrosocarboxylate and thea-nitrocarboxylate as substrates for the microsomal enzymesystem. a-Nitrosocarboxylates decarboxylate to oximes.a-Nitrocarboxylates are labile in aqueous solutions at phys-iological pH and decarboxylate to aci-nitro compounds (18,19). According to this reaction, the aci-tautomer of the nitrocompound is the enzymatically active species. aci-Nitro

R-CH-cOOi - R-CH-COOH R-CH-COOHNH2 NHOH NO2

compounds are in equilibrium with their parent nitro com-pounds. The equilibrium favors the nitro compound (19, 20),and the observed accumulation of the nitro compound withN-hydroxytyrosine as a substrate may reflect tautomeriza-tion of the aci-nitro compound released from the active siteon the microsomal enzyme system. The involvement of theaci-nitro compound as the enzymatically active specieswould explain the low metabolic activity observed uponadministration of the parent nitro compound (12).The almost quantitative incorporation of ['8O]oxygen into

the oxime with N-hydroxytyrosine as substrate (Table 1)precludes the possibility of free rotation around the C-Nbond of the a-nitrocarboxylate ion. An equilibrium between1-nitro-2-(p-hydroxyphenyl)ethane with free rotation aroundthe C-N bond and the aci-nitro tautomer during the enzy-matical conversion to the oxime is similarly excluded. Theseconclusions are in agreement with the earlier reported strongchanneling of the conversion of tyrosine to p-hydroxyphe-nylacetaldehyde oxime as demonstrated by double-labelingexperiments (17). The fixed orientation of the oxygen atomsintroduced by the successive N-oxidation reactions may beaccomplished by hydrogen bonds, metal chelation, or thepresence of a positively charged amino acid residue providedby the active site of the enzyme.

Alternative routes for the conversion of N-hydroxyty-rosine to p-hydroxyphenylacetaldehyde oxime are lesslikely. The involvement of an a-aci-nitrocarboxylic acid canbe excluded since this compound lacks the a-hydrogen atomoftyrosine. Experiments have shown that this hydrogen atomis retained when tyrosine is converted to p-hydroxyphenyl-acetaldehyde oxime (4). An alternative route would involveinitial decarboxylation of N-hydroxytyrosine to produceN-hydroxytyramine, which by an N-oxidation reaction maybe converted into the nitro compound. Since the sorghummicrosomal enzyme system does not utilize N-hydroxy-tyramine as a substrate and since N-hydroxytyramine is notlabeled in trapping experiments (3), this route is consideredunlikely.Apathway involvingdehydrogenationofN-hydroxy-tyrosine to 3-(p-hydroxyphenyl)-2-nitrosopropionate andsubsequent decarboxylation to produce the oxime has earlierbeen proposed (4, 21). This pathway is not consistent eitherwith the dioxygen stoichiometry data (12) or with the quan-titative incorporation of an [18O]oxygen atom from dioxygeninto p-hydroxyphenylacetaldehyde oxime when N-hydroxy-tyrosine is used as substrate as reported in this study. Theconversion of tyrosine to p-hydroxyphenylacetaldehyde ox-ime is therefore concluded to involve two N-hydroxylationsand a two-electron oxidation with an N-hydroxyamino acidand an a-nitrocarboxylic acid as intermediates. The a-nitro-carboxylic acid decarboxylates to produce the aci-nitro com-pound, which is stereoselectively reduced to an oxime. Thedata presented exclude the previously suggested positioningof the nitro compound as an intermediate between the oxime

R-csH-- Cyanogenic glucosides

|77,H NO

,H

NOH

R-CH-COOH NR-CH-OOH R-CH-COOH - 9~~4H2 NHOH NO2 -

Glucosinolates

FIG. 3. Comparison of the biosynthetic pathways for cyanogenic glucosides and glucosinolates with aci-nitro compounds as the branchingpoint.

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and the nitrile in the biosynthesis of cyanogenic glucosides(22-24). The biosynthetic studies indicate that the conversionof the nitro compound to the oxime is irreversible (12).

a-Nitrocarboxylic acids have never been isolated frombiological material. In 1968, Ettlinger and Kjaer (25) hypoth-esized that these might be intermediates in the biosynthesisof glucosinolates. Recently, L-nitrosuccinate has been re-ported as an intermediate in the biosynthesis of 3-nitropro-pionate from L-aspartate in Penicillium atrovenetum (26).Due to the lability of the a-nitrocarboxylic acid, the biosyn-thetic experiments were carried out by administration of thestable diethyl ester, reasoning that hydrolysis in vivo wouldrelease the free a-nitrocarboxylic acid within the fungal cells(26). The conversion of L-aspartate to 3-nitropropionateproceeds with both oxygens of the nitro group being derivedfrom dioxygen (27) and with retention ofthe a-hydrogen atom(26). These data would support a pathway analogous to thathere reported for the biosynthesis of dhurrin.

In the biosynthesis of glucosinolates (28), an aci-nitrocompound has been suggested as an intermediate betweenthe oxime and the S-alkylthiohydroximic acid (29). Theinvolvement of an aci-nitro compound in this pathway isattractive due to the possibility of forming the S-alkylthio-hydroximic acid by a nucleophilic attack on the a-carbonatom of the aci-nitro compound and a subsequent elimination(25). The suggestion was supported by the demonstration of1-nitro-2-phenylethane as a precursor of benzylglucosinolatein Tropaeolum majus (29). Trapping experiments where'4C-labeled phenylacetaldehyde oxime was fed to T. majus inthe presence of unlabeled nitro compound resulted in theproduction of '4C-labeled 1-nitro-2-phenylethane (29). Otherexperiments have shown that oximes are precursors forglucosinolates (30, 31) and that amino acids are precursors forthe oximes in glucosinolate-containing plants (32). Theseresults have led to the proposal that the oxime and theaci-nitro compound are intermediates in the biosynthesis ofglucosinolates (25) and that the aci-nitro compound is posi-tioned after the oxime (29). If these conclusions are correct,the position of the aci-nitro compound and the oxime asintermediates in the biosynthesis of cyanogenic glucosidesand glucosinolates is inverted. We do not favor this hypoth-esis. Criteria for the establishment of a compound as a trueintermediate include the demonstration ofenzymatic produc-tion and utilization of the compound (32, 33), but conclusionsbased solely on biosynthetic experiments become ambiguousifthe compound tested is in an enzyme-catalyzed or chemicalequilibrium with a true intermediate (32, 33). On the basis ofthe results presented here on the biosynthesis of cyanogenicglucosides, we speculate that the observed production of1-nitro-2-phenylethane from phenylacetaldehyde oxime andthe demonstrated trapping of oximes in feeding studies withamino acids represents side reactions in relation to thebiosynthetic pathway for glucosinolates. We propose that theaci-nitro compound is the branching point between the twobiosynthetic pathways (Fig. 3). Thus in plants producingcyanogenic glucosides, the aci-nitro compound is enzymat-ically reduced to the E-oxime, whereas in plants producingglucosinolates, the aci-nitro compound is converted to anS-alkylthiohydroximic acid by nucleophilic attack from asulfhydryl compound.

We thank Hanne Linde Nielsen and Inga Olsen for excellenttechnical assistance, Dr. J0rgen 0gaard Madsen for recording themass spectra, Profs. Anders Kjaer and Peder Olesen Larsen for

helpful comments about the manuscript, and Dr. Jim Foster (SeedtecInternational Inc., Hereford, TX) for continuously supplying thesorghum seeds. The work was partially supported by the DanishAgricultural and Veterinary Research Council, the Danish Interna-tional Development Agency, the Danish Government Program forBiotechnology Research, the Carlsberg Foundation, the RockefellerFoundation, and the Commission of the European CommunitiesScience and Technology for Development.

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