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Purification and Characterization of the Higher Plant Enzyme L-Canaline Reductase (L-Canavanine Catabolism/Plant Nitrogen Metabolism/Leguminosae) GERALD A

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Purification and Characterization of the Higher Plant Enzyme L-Canaline Reductase (L-Canavanine Catabolism/Plant Nitrogen Metabolism/Leguminosae) GERALD A 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.
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