Proc. Natd. Acad. Sci. USA Vol. 89, pp. 1780-1784, March 1992 Biochemistry Purification and characterization of the higher plant enzyme L-canaline reductase (L-canavanine catabolism/plant nitrogen metabolism/Leguminosae) GERALD A. ROSENTHAL T. H. Morgan School of Biological Sciences, University of Kentucky, Lexington, KY 40506 Communicated by John S. Boyer, December 3, 1991 (receivedfor review April 20, 1991)

ABSTRACT A newly discovered enzyme, L-canaline re- oglutaric acid to generate stoichiometrically a canaline-2- ductase (NADPH:L-canaline oxidoreductase, EC 1.6.6.-), has oxoglutaric acid oxime (5). Canaline also reacts readily with been isolated and purified from 10-day-old leaves of the jack the pyridoxal phosphate moiety of vitamin B6-containing bean Canavalia ensiformis (Leguminosae). This higher plant is enzymes to form a stable, covalently linked complex (6, 7). representative of a large number of legumes that synthesize In vitro analysis of canaline interaction with homogeneous L-canavanine, an important nitrogen-storing nonprotein amino ornithine aminotransferase (ornithine-oxo-acid aminotrans- acid. Canavanine-storing legumes contain arginase, which ferase; L-ornithine:2-oxo-acid aminotransferase, EC hydrolyzes L-canavanine to form the toxic metabolite L-cana- 2.6.1.13) reveals its marked ability to form an oxime complex line. Canaline reductase, having a mass of =167 kDa and with and thereby inhibit this pyridoxal phosphate-dependent composed of 82-kDa dimers, catalyzes a NADPH-dependent enzyme (8). As little as 10 nM canaline causes a significant reductive cleavage of L-canaline to L-homoserine and ammo- reduction in ornithine aminotransferase activity (9). nia. This is the only enzyme known to use reduced NADP to Canavanine-storing legumes can accumulate high levels of cleave an O-N bond. Canaline reductase performs at least this nonprotein amino acid; for example, canavanine can three important functions for canavanine-synthesizing le- account for up to 13% of the total dry matter of the seed of gumes. First, it detoxifies canaline. Second, it increases by certain legumes (9). To support their nitrogen metabolism, one-halfthe overall yield ofammoniacal nitrogen released from these plants use their arginase to release the stored nitrogen canavanine. Third, it permits the carbon skeleton of canava- of canavanine as urea (10); canaline is a toxic by-product of nine, a secondary plant metabolite, to support vital primary this catabolic reaction. Analysis of these canavanine-storing metabolic reactions. legumes discloses the presence of only trace amounts of canaline (5, 11). To avoid the toxic effects of canaline, these plants have evolved a mechanism to efficiently catabolize the L-Canavanine [L-2-amino-4-(guanidinooxy)butyric acid] is an appreciable canaline formed from canavanine. L-arginine analog that occurs in at least 1500 legumes (1). This Investigation of the canavanine-producing jack bean nonprotein amino acid can be the most abundant free amino Canavalia ensiformis (Leguminosae) has resulted in the acid of the plant (2). Arginase (L-arginine amidinohydrolase, isolation of an enzyme that used NADPH to reductively EC 3.5.3.1) appears to be distributed universally in these cleave the oxygen-nitrogen bond of canaline irreversibly to canavanine-storing legumes (2). Since canaline is a product of yield homoserine and ammonia. arginase-mediated hydrolysis of L-canavanine, all of these legumes are a potential source of L-canaline [L-2-amino-4- H2N-0--CH2-CH2--CH(NH2)COOH (aminooxy)butyric acid]. L-canaline H2N-C(NH2)=N--O-CH2-CH2-CH(NH2)COOH HO-CH2-CH2-CH(NH2)COOH + NH3 L-canavanine L-homoserine ammonia H2N-O-CH2--CH2-CH(NH2)COOH This communication describes the purification and charac- L-canaline terization of the enzyme L-canaline reductase (CR; NAD- PH:L-canaline oxidoreductase, EC 1.6.6.-), which not only + H2NC(==O)NH2 detoxifies canaline but also supports the nitrogen metabolism urea of the plant. Canaline is a toxic natural product that elicits potent MATERIALS AND METHODS insecticidal properties in canaline-sensitive insects. For ex- ample, consumption of a 2.5 mM canaline-containing artifi- Substrate Preparation. L-Canavanine (free base) was iso- cial diet by larvae of the tobacco hornworm Manduca sexta lated from acetone-defattedjack bean seeds by ion-exchange (Sphingidae) results in massive developmental aberrations in chromatography and purified by repetitive crystallization the pupae and adults that emerge from the larvae. Most ofthe (12). L-Canaline (free base) was prepared from L-canavanine canaline-treated larvae perish attempting larval-pupal meta- by the method of Rosenthal (13). morphosis (3). Canaline also induces neurotoxic effects in the L-[U-14C]Canaline was synthesized from commercially adult moth (4). prepared L-[U-14C]homoserine (Amersham; 1.48 GBq/ The toxic action of canaline results from its reactivity with mmol). The radiochemical synthesis involved the successive the carbonyl group ofaldehydes or certain keto acids to form synthesis of L-2-[(benzyloxycarbonyl)amino]-4-hydroxybu- oximes. For example, canaline reacts chemically with 2-ox- tyric acid, L-2-[(benzyloxycarbonyl)amino]-4-butyrolactone, benzyl L-2-[(carbobenzyloxy)amino]-4-hydroxybutyrate, and benzyl The publication costs of this article were defrayed in part by page charge L-2-[(carbobenzyloxy)amino]-4-[(p-tolylsulfo- payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: CR, L-canaline reductase. 1780 Downloaded by guest on September 30, 2021 Biochemistry: Rosenthal Proc. Natl. Acad. Sci. USA 89 (1992) 1781 nyl)oxy]butyrate. The aminooxy function was introduced dissolved in deionized water, and the process was repeated with the addition of benzohydroxamic acid to form benzyl twice. Finally, the '4C-bearing residue was taken up in a L-2-[(carbobenzyloxy)amino]-4-(benzamidooxy)butyrate. minimum amount of deionized water and reacted with suffi- This compound was deprotected by refluxing with l9o cient 2-oxoglutaric acid to convert any unreacted ['4C]cana- (wt/vol) ethanolic HCl followed by refluxing with 3 M HCl to line to its radiolabeled canaline-2-oxoglutarate oxime. Unlike form L-[U-14C]canaline (14). Scintillation medium (Ecolume) canaline, this oxime can be quantitated since it is stable to the was purchased from ICN. Sigma supplied the biochemicals. buffers used in automated amino acid analysis. All other chemicals were obtained from Aldrich. The "'C-bearing effluent was subjected to automated amino Canaline-Linked, Cyanogen Bromide-Activated Sepharose. acid analysis in which the column effluent was collected A suspension ofSepharose (40-190 jm) in deionized water was without reacting with ninhydrin. The column effluent was centrifuged at 600 x g for 10 min to pack the gel. Ten then assayed by liquid scintillation spectroscopy. Radiola- milliliters (settled gel volume) of the gel was suspended in 10 beled homoserine (retention time, 32 min) accounted for 98% ml of deionized water, taken to pH 10.5-11 with 4 M NaOH, ofthe 'IC ofthe column effluent not residing in ["'Cicanaline- and stirred mechanically in a well ventilated hood with 1.0 g 2-oxoglutarate oxime (retention time, 14 min). of cyanogen bromide dissolved in 15 ml of 1-methyl-2- To verify the identity of homoserine, a portion of the pyrrolidinone. The pH was maintained between 10 and 11 for radiolabeled effluent from the Dowex 50 column was refluxed 12 min. The cyanogen bromide-activated gel was transferred with 9%o (wt/vol) ethanolic HCO for 90 min. After removing to a sintered glass funnel and washed with 10 vol of ice-cold the solvent by rotary evaporation in vacuo, the residue was 100 mM sodium carbonate/bicarbonate buffer (pH 9.5) (buff- allowed to in vacuo at for an 30 min after er A). dry 550C additional The activated gel in buffer A was treated with 100 mg of solvent removal. Deionized water was added to the residue L-canaline and agitated gently overnight at 230C. After wash- and the drying process was repeated twice. The residue was ing the canaline-linked Sepharose with buffer A, it underwent then dissolved in 3 M HCl and refluxed for 90 min at 115TC. reaction with 3 vol of 1 M 2-aminoethanol (pH 9.5) for 60 min Afterwards, the HCI was removed by exhaustive rotary at 23°C. After transferring the treated gel to a sintered glass evaporation in vacuo and the residue was dissolved in funnel, it was washed thoroughly with buffer A and stored in deionized water, taken to pH 3.5 with 1 M ammonia, and 50 mM sodium acetate (pH 4.0) with a few crystals of sodium placed on a column (20 x 75 mm) of Dowex 50 (NH4+). The azide at 4°C. column was washed with 0.7 liter of deionized water and Enzyme Assay. Crude CR activity was determined by developed with 0.5 liter of 200 mM ammonia. The column measuring the degradation of L-canaline to L-homoserine in effluent was concentrated by rotary evaporation in vacuo. the presence of a reduced NADP-regenerating system. The The above procedures convert homoserine stoichiometri- assay mixture (1.0 ml) consisted of 15 mM L-canaline (pH cally to homoserine lactone; unlike homoserine, the latter 7.3), 100 mM sodium Tricine (pH 7.3), 2.5 mM NADP, 25 mM compound is basic. Free homoserine, unreacted canaline, glucose 6-phosphate, 150 ,ug of glucose-6-phosphate dehy- neutral compounds, and acidic compounds cannot bind to drogenase (300 units per mg of protein), and no more than this resin in the NH4+ form. Homoserine lactone is converted 0.065 unit of CR. All assays were conducted in triplicate at to homoserine in situ when the column is developed with 200 37°C for 60 min. The reaction was terminated by the addition mM ammonia. All of the 14C adhering to the exhaustively of 2.0 ml of 3.5 M perchloric acid, neutralized with 2.0 ml of washed column eluted with 200 mM ammonia. Automated 3.5 M KOH and placed on ice for several minutes. After amino acid analysis of the column effluent established that clarifying the assay mixture by centrifugation, the superna- homoserine was the sole "4C-bearing compound. tant solution was assayed colorimetrically for the disappear- Identification of Ammonia. Ammonia production resulting ance of canaline. Controls consisted of appropriate reaction from CR-catalyzed reduction of canaline was established by mixtures to which boiled enzyme was added. monitoring the oxidation of NADH in the presence of glu- Purified CR was assayed by monitoring the decrease in tamic acid dehydrogenase and 2-oxoglutaric acid as de- absorbance at 340 nm resulting from the oxidation of scribed by Tabor (16). NADPH. The assay mixture (1.0 ml) included 15 mM L-cana- Competitive Inhibitor Kinetics. The Ki value for the com- line (pH 7.3), 100 mM sodium Tricine (pH 7.3), 0.1 mM petitive inhibitors aminooxyacetic acid and hydroxylamine NADPH, and no more than 0.05 unit of CR. Assays were was determined from a series of Lineweaver-Burk reciprocal conducted at 23°C in a Gilford response recording spectro- plots of 1/v vs. 1/[S] in the presence of various inhibitor photometer that determined the absorbance 10 times per min. concentrations. The slope of the reciprocal plots when in- One unit of enzyme catalyzed the reduction of 1 ,mol of hibitor is present can be represented as: canaline per min under the described reaction conditions. Colorimetric Assay. Canaline depletion was determined slope = Km/[Vmax.Ki][I] + Km/Vmax. colorimetrically; canaline was carbamoylated with cyanate to generate ureidohomoserine prior to color development (15). The slope obtained from each of the reciprocal plots was The neutralized enzyme assay mixture (0.5 ml) was diluted then plotted as a function of the inhibitor concentration. The with an equal vol of 100 mM sodium acetate buffer (pH 4.0) x intercept of the resulting curve = -K,. and treated with 1.0 ml of 1:1 (vol/vol) oxime/semidine Protein Assay. Protein was determined in crude prepara- reagent and then 0.5 ml of concentrated sulfuric acid. The tions by the method of Lowry et al. (17). The absorbance of tubes were placed into boiling water for exactly 2.5 min; the purified CR was determined with the exhaustively dialyzed resulting chromogen was read immediately at 542 nm (15). enzyme that was dried to constant weight in vacuo at 100°C. Identification of Homoserine. The standard enzyme assay A 0.1% (wt/vol) solution of CR had an A280 of 1.05. mixture, containing a 10-fold excess of reagents and 0.37 Polyacrylamide Gel Electrophoresis. CR was analyzed by MBq of L-[U-14C]canaline, underwent reaction at 37°C for 4 polyacrylamide gel electrophoresis under nondenaturing h. Afterwards, the reaction mixture was deproteinized as conditions by the method of Davis (18). The protein, treated described above, taken to pH 3.5 with 2 M HCI, and applied with SDS under reduced conditions for 3 min at 100°C, was to a column (20 x 75 mm) of Dowex 50 (HW). After washing also analyzed by the method of Laemmli (19). All gels were the column with 1 liter of deionized water, it was developed stained with 0.5% (wt/vol) Coomassie blue. Commercially with 0.5 liter of 150 mM ammonia. The column effluent was prepared proteins (ranging from 14 to 181 kDa) served as the concentrated by rotary evaporation in vacuo, the residue was standards (Sigma SDS-6 and SDS-7 standards). Downloaded by guest on September 30, 2021 1782 Biochemistry: Rosenthal Proc. Natl. Acad. Sci. USA 89 (1992)

Amino Acid Analysis. CR samples were hydrolyzed in 6 M time to 1-2 ml; 0.2-ml fractions were stored at -60TC (Table HCl with 0.02% (vol/vol) 2-mercaptoethanol and 0.01% 1). Enzyme assays were conducted with protein purified (wt/vol) phenol for 24 h at 100°C. The amino acid composi- through this step. tion of the hydrolyzate was established by ion-exchange 111gb-Performance Liquid Chromatography. Traces ofcon- chromatography using a Dionex D-300 automated amino acid taminant protein were further removed by injecting the analyzer equipped with a lithium citrate physiological buffer concentrated affinity column effluent (200 1.l) into a reverse- system and ninhydrin detection. Tryptophan and half-cystine phase C18-WP column (Bakerbound) and the protein was were determined as described (20). Amino acids were stan- eluted over a period of 40 min with a linear gradient of dardized with Pierce amino acid standards. Peak area was solution A [0.1% (vol/vol) aqueous trifluoroacetic acid] and determined by a Spectra physics model 4270 automated area solution B [80%o acetonitrile and 20% (vol/vol) solution A]. integrator. Fractions of 1.0 ml were collected at 1-min intervals. The Absorbance. Absorbance spectra were obtained with a effluent was monitored at 280 nm with an SSI model 500 solution (0.5 mg/ml) of purified enzyme that was dialyzed variable-wavelength spectrophotometer. overnight against standard buffer. A sample of the dialyzed CR is a labile enzyme that loses activity when maintained enzyme was reacted with 30 ,uM NADPH. Absorbance was on ice and it is highly unstable in the absence of protective measured with a Gilford response spectrophotometer. mercaptans. CR maintained in 50 mM buffer lost virtually all Kinetic Analysis. Apparent Km values were taken from of its activity overnight at 3YC; in the presence of suitable modified Lineweaver-Burk plots determined by regression protecting mercaptans, this loss was reduced by about one- analysis. Lines having an r value of <0.999 were discarded. half. Purified CR was stored as a pellet under liquid-saturated Turnover number was calculated from Vma. data obtained on ammonium sulfate at -600C; the frozen enzyme was used these plots and was based on a molecular mass of 168 kDa. within a few weeks of its preparation. RESULTS Characterization of CR. Characterization of the reaction. Preparation of CR. Jack bean, C. ensiformis [Legumino- Analysis of the standard enzyme assay mixture containing sae], seedlings were greenhouse-grown from seeds for 10 ['4C]canaline revealed that homoserine was the sole radio- days as described (21). Frozen 10-day-old jack bean leaves labeled reaction product. Separate analysis established am- (75 g) were ground with a Sorvall Omni mixer at full power monia as another reaction product of canaline reduction. for 30 sec with 300 ml of buffer. Unless otherwise indicated, Isolation and identification of these reaction products were the buffer was 50 mM sodium Tricine (pH 7.3) containing conducted as described. This reaction appears to be irrevers- 0.1% (vol/vol) 2-mercaptoethanol and 1 mM dithiothreitol. ible as reaction of CR with L-homoserine and such nitrogen This buffer provided the most stable storage condition while donors as ammonia, glutamine, asparagine, or hydroxyl- yielding maximum enzymatic activity. amine failed to produce any detectable canaline. The limit of Purifiation of CR. The jack bean leaf homogenate was canaline detectability was 10 nmol. expressed through cheesecloth and then centrifuged for 20 NADPH served as a highly effective reductant for the min at 12,500 x g. A total of 225 g of frozen leaves were conversion of canaline to homoserine. Spectral analysis of processed. The supernatant solution, combined from the CR showed that the reduced coenzyme could bind to the three grindings, was taken to 55% saturation by the addition enzyme without canaline being present (Fig. 1); NADPH was of a liquid, saturated ammonium sulfate solution (pH 7.6) and present in the blank. NADH did not function as an effective allowed to sit at 4°C for at least 90 min. The turbid solution agent for canaline reduction. ATP/Mg2' did not enhance the was centrifuged at 12,500 x g for 20 min. The pellet was formation of homoserine from canaline. dissolved in a minimum amount of buffer and dialyzed Mass. The molecular mass ofthe CR subunit was obtained overnight against 2 x 2 liters of the same buffer. Centrifu- by SDS/polyacrylamide gel electrophoresis with proteins of gation was used to remove protein that precipitated during known mass used as the standard. This procedure yielded a dialysis. single band with an apparent molecular mass of 82 ± 3 kDa. DEAE-Cellulose Chromatography. The dialyzed enzyme The molecular mass ofjack bean CR was determined to be solution was diluted with an equal volume ofdeionized water 170 ± 5 kDa by polyacrylamide gel electrophoresis under immediately before its application to a column (28 x 375 mm) nondenaturing conditions. Molecular mass evaluation by of Whatman DEAE-cellulose (DE32) equilibrated with 20 Sephadex G-200 chromatography yielded a value of 167 ± 6 mM buffer. The column was washed with 200 ml of 75 mM kDa. These data suggest thatjack bean CR exists as a dimer buffer. composed of two 82-kDa units. CR was obtained by developing the washed column with a Automated amino acid analysis. Automated amino acid linear gradient consisting of 100 ml of 100 mM buffer and an analysis of CR disclosed a high proportion of aspartate/ equal volume of 400 mM buffer. The effluent, at a flow rate asparagine and glutamate/glutamine in the protein. Aromatic of 1.5 ml-min-', was collected at 2-min intervals. Assays residues were limited but the proportion of hydrophobic were conducted with 100 Jul of each fraction; the five most residues was high. There were 12 cysteine residues (Table 2). active fractions were pooled. Ellman titration of the native protein disclosed 8 sulfhydryl G-200 Sephadex Chromatography. The concentrated efflu- ent was applied to a column (10 x 420 mm) ofSephadex G-200 Table 1. Purification of CR from C. ensiformis equilibrated with buffer. Fractions (1 ml) were collected at Total Specific 12-min intervals. Assays were conducted with 50 ,ul of each activity, Protein, activity, Yield, fraction; the six most active fractions were pooled, concen- Fraction units mg units/mg' % trated as described above, and stored at -60'C. Affinity Chromatography. The concentrated enzyme solu- Homogenate 3000 460 6.5 tion (1.0 ml) was applied to a column (7 x 40 mm) of Ammonium sulfate canaline-linked Sepharose equilibrated with 20 mM buffer. (0-55% saturation) 2875 135 21.3 96 The column was washed with the same buffer and developed DEAE-cellulose 860 30 28.7 29 with 200 mM buffer containing 100 mM KCI; 1.0-ml fractions Sephadex G-200 10 were collected. After concentrating the three most active chromatography 290 6.8 42.6 fractions by Amicon filtration using a 10-ml cell, the enzyme Affinity chromatography 165 0.27 611.1 3 was diluted twice with 50 mM buffer and concentrated each Experimental details are provided in the text. Downloaded by guest on September 30, 2021 Biochemistry: Rosenthal Proc. Natl. Acad. Sci. USA 89 (1992) 1783

0.7 80 0.6

J 0.5 z E 60 m< 0.4 E / 160 en0 0.3 C m 40 0 > 120F-s I- [ 0.2 0 7 40 0.1 20 -2 0 2 4 6 8 10 280 320 360 400 440 480 [CANALINE] (mM) WAVELENGTH (nm) 0 2 4 6 8 10 FIG. 1. Absorbance spectra of CR. The absorbance spectrum of CR (e) and enzyme reacted with 30MuM NADPH (v) were determined [CANALINE] (mM) as described in the text. FIG. 2. The course of L-canaline reduction by CR. (Inset) Kinetic groups; this value increased to no more than 10 when CR was analysis of CR. Reduction of canaline and determination of the apparent Km value were conducted as described in the text. Velocity treated with 6 M guanidine. is in nmol/min. Kinetic parameters. Kinetic analysis of CR indicated that the apparent Km values for L-canaline and NADPH were 0.76 CR is a unique enzyme in two ways. First, it is the only (Fig. 2) and 0.16 mM, respectively. The highest specific enzyme able to metabolize canaline, which cannot function activity CR gave a turnover number of 1.224 x 104 ,umol per with ornithine. Previous reports of enzymes capable of min per umol of CR. metabolizing canaline were limited strictly to proteins for Inhibitor studies. Kinetic analysis ofCR revealed that the Ki which canaline served as a substrate only by virtue of its values for the competitive inhibitors aminooxyacetic acid and structural analogy to ornithine. For example, ornithine car- hydroxylamine were 4.9 and 7.9 mM, respectively (Fig. 3). bamoyltransferase (carbamoyl-phosphate:L-aspartate car- Sulflydryl inactivation. Treatment of CR with N-ethylmale- bamoyltransferase, EC 2.1.3.3) not only carbamylates orni- imide established the sensitivity of the enzyme to this sulfhy- thine to citrulline but also carbamylates canaline to form dryl-group inhibitor. Exposure of CR to 1 mM N-ethylmaleim- O-ureido-L-homoserine (22, 23). In contrast, CR metabolizes ide for 20 min inactivated the enzyme (Fig. 4). Even exposure canaline but in vitro experiments with CR and ornithine to 1 kLM inhibitor severely affected CR activity (Fig. 4). reveal no demonstrable catabolism of ornithine. Thus, this DISCUSSION enzyme is not reactive with ornithine. Second, CR is the only presently known enzyme able to use NADPH to reductively CR, purified from the leaves of C. ensiformis, mediates an cleave an O-N bond. NADPH-dependent, stoichiometric reduction of L-canaline Canaline reductase has a moderately high affinity for to L-homoserine and ammonia. CR has a molecular mass of canaline, as judged by the apparent Km value for canaline of about 167 kDa and is composed of dimers with a mass of 82 0.76 mM. This substrate affinity would facilitate catabolism kDa. It is a labile enzyme that rapidly loses its catalytic of low levels of canaline and may contribute significantly to activity if not protected by mercaptans. the paucity ofcanaline in canavanine-storing plants (5). Many leguminous plants store massive amounts of canavanine and of C. CR Table 2. Amino acid composition ensiformis they also possess appreciable arginase activity (2). Canaline Amino acid Mol % Residues per mol Asx 10.42 149 6 Thr 4.32 70 Ser 4.20 79 2 5 Glx 16.40 209 Pro 4.68 79 u4 Gly 3.10 89 Ala 4.21 97 Cys 0.38 12 0. Val 6.35 105 Met 4.24 53 Ile 9.26 134 Leu 8.63 125 Tyr 2.69 27 -8 -6 -4 -2 0 2 4 6 8 10 Phe 2.87 32 Lys 5.95 76 [l] (mM) His 3.85 46 Arg 6.29 66 FIG. 3. Competitive inhibitor kinetic analyses of CR. The K; and acid (e) were 2.16 19 values for hydroxylamine (v) aminooxyacetic Trp determined from the slope value obtained from a series ofplots of 1/v Total, 1467 residues; mass, 163,875 Da. Experimental details are vs. 1/[S] at the indicated inhibitor concentration. See text for provided in the text. additional experimental details. Downloaded by guest on September 30, 2021 1784 Biochemistry: Rosenthal Proc. Natl. Acad. Sci. USA 89 (1992) primarily part of the organism's secondary metabolism, to 100 support the primary metabolic reactions of canavanine- containing legumes. 80 I gratefully acknowledge the financial support of the National Science Foundation (Grant DK17322) and the Graduate School ofthe 60 University of Kentucky. I-- 5: 1. Bell, E. A., Lakey, J. A. & Polhill, R. M. (1978) Biochem. I-- 40 Syst. Ecol. 6, 201-212. 2. Rosenthal, G. A. (1974) J. Exp. Bot. 25, 609-613. 3. Rosenthal, G. A. & Dahlman, D. L. (1975) Comp. Biochem. 20 Physiol. 52A, 105-108. 4. Kammer, A., Dahlman, D. L. & Rosenthal, G. A. (1978) J. Exp. Biol. 75, 123-132. 0 5. Rosenthal, G. A., Berge, M. & Bleiler, J. (1989) Biochem. Syst. 0 20 40 60 Ecol. 17, 203-206. TIME (min) 6. Rosenthal, G. A. (1981) Eur. J. Biochem. 114, 301-304. 7. Beeler, T. & Churchich, J. E. (1976) J. Biol. Chem. 251, FIG. 4. N-Ethylmaleimide inactivation of CR. CR (1.2 ,ug) un- 5267-5271. derwent reaction with N-ethylmaleimide for the indicated time in 0.1 8. Rosenthal, G. A. & Dahlman, D. L. (1990) J. Biol. Chem. 265, ml of standard buffer. The treated enzyme was assayed for activity 868-873. 9. Rosenthal, G. A., Dahlman, D. L. & Janzen, D. H. (1976) as described in the text. e, v, v, o, and *, 0, 10-6, 10-5, 10-4, and 10-3 M N-ethylmaleimide, respectively. Science 192, 256-258. 10. Rosenthal, G. A., Berge, M., Ozinskas, A. & Hughes, C. H. must form in these plants. CR provides a highly effective (1988) J. Food Agr. Chem. 36, 1159-1163. 11. Rosenthal, G. A. & Berge, M. (1989) J. Food Agr. Chem. 37, means of detoxifying this potent antimetabolite. 591-595. Higher plants need to conserve nitrogen because this 12. Rosenthal, G. A. (1977) Anal. Biochem. 77, 147-151. element is often a rate-limiting nutrient for growth (22). CR 13. Rosenthal, G. A. (1973) Anal. Biochem. 51, 354-361. supports the vital nitrogen metabolism of legumes such as 14. Ozinskas, A. & Rosenthal, G. A. (1986) J. Org. Chem. 51, jack bean in two important ways. First, it provides a means 5047-5050. to release the nitrogen stored in the aminooxy moiety of 15. Rosenthal, G. A. & Thomas, A. (1984) Anal. Biochem. 140, canaline as ammonia. Thus, plants that can convert canava- 246-249. 16. Tabor, C. W. (1970) Methods Enzymol. 16, 955. nine to canaline and then canaline to homoserine gain all 17. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. three of the nitrogens of the guanidinooxy group of canava- (1951) J. Biol. Chem. 193, 265-275. nine (directly as well as through urea hydrolysis). Second, 18. Davis, B. J. (1964) Ann. N. Y. Acad. Sci. 121, 404-427. homoserine is a vital precursor in the sulfur metabolism of 19. Laemmli, U. K. (1970) Nature (London) 227, 680-685. legumes (23). Higher plants phosphorylate homoserine to 20. Rosenthal, G. A., Dahlman, D. L. & Robinson, G. W. (1977)J. phosphohomoserine; the latter functions in the biosynthesis Biol. Chem. 252, 3679-3683. of such critically important sulfur-containing metabolites as 21. Rosenthal, G. A. (1970) Plant Physiol. 46, 273-276. and methionine CR not 22. Miflin, B. J. & Lea, P. J. (1980) in The Biochemistry ofPlants: cystathionine, homocysteine, (23). A Comprehensive Treatise, eds. Stumpf, P. K. & Conn, E. E. only functions in canaline detoxification, thereby contribut- (Academic, New York), pp. 169-202. ing to the low natural abundance ofcanaline, but it also serves 23. Giovanelli, J., Mudd, S. H. & Datko, A. H. (1980) in The as an important bridge between canavanine and homoserine. Biochemistry of Plants: A Comprehensive Treatise, eds. This is a particularly important function because CR provides Stumpf, P. K. & Conn, E. E. (Academic, New York), pp. a means for a metabolite such as canavanine, which is 454-506. Downloaded by guest on September 30, 2021