Proc. Natl. Acad. Sci. USA Vol. 94, pp. 2255–2260, March 1997 Biochemistry

The biochemical basis for L-canavanine tolerance by the tobacco budworm Heliothis virescens (Noctuidae) (allelochemical detoxification)

COROMOTO MELANGELI*, GERALD A. ROSENTHAL†‡, AND DOUGLAS L. DALMAN§

*Universidad Central de Venezuela, Centro de Bioquimica Nutricional, Maracay, Venezuela; †Laboratory of Biochemical Ecology and §Department of Entomology, University of Kentucky, Lexington, KY, 40506

Communicated by William Bowers, University of Arizona, Tucson, AZ, December 23, 1996 (received for review February 5, 1996)

ABSTRACT The tobacco budworm, Heliothis virescens tained on a 2.5 mM canavanine-containing diet develop (Noctuidae), a destructive insect pest, is remarkably resistant massive developmental aberrations (1, 2). The appreciable to L-canavanine, L-2-amino-4-(guanidinooxy)butyric acid, an natural resistance of H. virescens to this protective allelo- arginine antimetabolite that is a potent insecticide for non- chemical instigated earlier studies to elucidate the biochem- adapted species. H. virescens employs a constitutive enzyme of ical basis for canavanine tolerance by H. virescens (7–9). the larval gut, known trivially as canavanine hydrolase (CH), These studies established that H. virescens actively metab- to catalyze an irreversible hydrolysis of L-canavanine to olizes canavanine under the control of a constitutive enzyme L-homoserine and hydroxyguanidine. As such, it represents a that has been isolated from the larval gut (8). Administration new type of hydrolase, one acting on oxygen–nitrogen bonds of L-[guanidinooxy-14C]canavanine to fifth instar H. virescens (EC 3.13.1.1). This enzyme has been isolated from the excised larvae disclosed that [14C]guanidine was the principal in vivo gut of H. virescens and purified to homogeneity; it exhibits an larval radiolabeled canavanine catabolite (9). Subsequent in 14 apparent Km value for L-canavanine of 1.1 mM and a turnover vivo studies employing L-[1,2,3,4- C]canavanine identified number of 21.1 ␮mol⅐min؊1⅐␮mol؊1. This enzyme has a mass L-[1,2,3,4-14C]homoserine as the preponderant radiolabeled of 285 kDa and is composed of two subunits with a mass of 50 catabolite (9). These studies of radiolabeled metabolite dis- kDa or 47.5 kDa. CH has a high degree of specificity for position implicated a larval reductase in the detoxification of L-canavanine as it cannot function effectively with either canavanine via homoserine and guanidine. L-2-amino-5-(guanidinooxy)pentanoate or L-2-amino-3- (guanidinooxy)propionate, the higher or lower homolog of L-canavanine, respectively. L-Canavanine derivatives such as methyl-L-canavanine, or L-canaline and O-ureido-L-homo- serine, are not metabolized significantly by CH.

L-Canavanine, the L-2-amino-4-(guanidinooxy)butyric acid structural analog of L-arginine synthesized by leguminous plants, is normally a potent insecticide (1, 2); it provides a More recent experiments revealed a larval gut reductase able formidable chemical barrier against predation by nonadapted to catalyze an NADH-dependent reduction of hydroxyguani- species (3). dine to guanidine (10). This finding suggested that canavanine was catabolized initially to homoserine and hydroxyguanidine by a hydrolase able to cleave the O–N bond of the guanidi- nooxy moiety of the substrate. While this metabolic ability has been observed in a soil-borne Pseudomonas (11), the respon- sible enzyme was not isolated nor has this metabolic capacity been described from an eukaryotic organism. This communication details purification and characteriza- tion of this canavanine-degrading enzyme, which we have In contrast, insects such as the bruchid beetle, Caryedes named canavanine hydrolase (CH). This protein mediates an brasiliensis and the weevil, Sternechus tuberculatus, which feed irreversible hydrolysis of L-canavanine to L-homoserine and exclusively on canavanine-containing seeds, are adapted to this hydroxyguanidine. potentially poisonous natural product (4, 5). Indeed, C. bra- siliensis employs L-canavanine to provide the nitrogen for the biosynthesis of many essential amino acids (5, 6). The tobacco budworm, Heliothis virescens (Noctuidae), exhibits a remarkable degree of tolerance to dietary cana- vanine as the larvae can be reared on a diet containing 300 mM canavanine, or nearly 40% nonprotein amino acid by dry weight, with only a marginal effect on larval growth and without eliciting discernible developmental aberrations (7). In contrast, tobacco hornworm larvae, Manduca sexta, main- In this reaction, CH functions as a hydrolase that cleaves an oxygen–nitrogen bond; this enzyme is the only protein known to demonstrate this ability. (12). As such it represents a new The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: CH, L-canavanine hydrolase; PCAF, pentacyanoammo- nioferrate; TFA, trifluoroacetic acid. Copyright ᭧ 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA ‡To whom reprint requests should be addressed at: 101 T. H. Morgan 0027-8424͞97͞942255-6$2.00͞0 Building, University of Kentucky, Lexington, KY 40506. e-mail: PNAS is available online at http:͞͞www.pnas.org. [email protected].

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type of hydrolase, one that acts on oxygen–nitrogen bonds (EC The column fractions were assayed for arginase activity by 3.13.1.1). treating 50 ␮l of sample with 25 mM L-arginine (pH 9.7), 1 mM MnCl2, and 50 mM sodium glycylglycine buffer (pH 9.7) in a MATERIALS AND METHODS final volume of 0.5 ml for 10 min at 37ЊC. The assay was terminated by transferring 100 ␮l of the reaction mixture to a Substrates. L-Canavanine free base was obtained by recrys- tube containing 2.9 ml of a solution of 1.25% (wt͞vol) ninhy- tallization of material isolated from a methanolic extract of drin in glacial acetic͞6 M phosphoric acid (4:1, vol͞vol) after Jack bean seed, Canavalia ensiformis (13). L-Canaline was the method of Chinard (20). After standing in boiling water for synthesized from canavanine by arginase-mediated hydrolysis 10 min, the tubes were inspected visually for the orange-red and subsequent isolation of the dipicric acid salt of canaline; chromogen produced by ornithine. The 45 most active frac- canaline free base was prepared via canaline sulfate (13). tions were pooled, concentrated to 20 ml by ultrafiltration with O-Ureido-L-homoserine was made from canaline by carbamy- an Amicon P-10 membrane, and stored as 1.0 ml samples at lation with KCNO (14). L-Homocanavanine was prepared Ϫ80ЊC. from L-2-amino-5-hydroxypentanoic acid, the higher homolog Every arginase preparation was evaluated for canavanine- of L-homoserine, as described (15). The lower homolog of degrading ability by reacting 100 ␮l of purified arginase with L-canavanine was synthesized from L-cycloserine by ring open- 25 mM L-canavanine, under the conditions of the CH assay, at ing followed by zinc-mediated guanidination with cyanamide 37ЊC for 7 h. In all instances, the above procedure provided (16). The methyl, ethyl, and butyl ester of canavanine were arginase of sufficient activity to degrade all canavanine as produced from the parent compound (15). Barium [14C]cy- determined by the PCAF colorimetric assay. anamide (2.15 GBq͞mmol) was synthesized by Amersham. All Protein Assay. Protein was determined in crude prepara- remaining reagents were secured from Sigma͞Aldrich. tions by the Bio-Rad protein assay mixture. The absorbance of Insects. H. virescens terminal instar larvae were reared in a homogeneous canavanine hydrolase was determined as de- continuous colony maintained at the University of Kentucky as scribed (21). A 0.1% solution (wt͞vol) had an A280 of 1.35. described (17). All insects were anesthetized by chilling on ice Amino Acid Evaluations. Canaline was monitored by a before gut collection. colorimetric procedure after its conversion to O-ureidohomo- Canavanine Hydrolase Assay. Because of their equivalent serine by reaction with 20 mM KCNO in 100 mM sodium basicity, hydroxyguanidine cannot be separated effectively acetate buffer (pH 4.5) (14). O-Ureidohomoserine was deter- from canavanine by ionic-exchange chromatography. More- mined directly by the same colorimetric procedure (14). Ho- over, hydroxyguanidine reacts with pentacyanoammoniofer- moserine was quantified by automated amino acid analysis rate (PCAF), the reagent that is also used for colorimetric employing a Dionex model D-300 automated amino acid analysis of canavanine, to generate a stable chromogen. The analyzer with lithium citrate gradient elution and ninhydrin detection at 570 nm. Homoserine eluted after 17 min, ammo- PCAF colorimetric assay is the only suitable reaction for nia eluted at 73 min, and canavanine after 105 min. colorimetric analysis of hydroxyguanidine, but the canava- Kinetic Analysis. The apparent K value, determined by nine–PCAF chromogen, produced from unreacted substrate, m regression analysis, was taken from Hanes–Woolf modification masks the hydroxyguanidine–PCAF chromogen. To overcome of Lineweaver–Burk plots. Lines having an r value less than this difficulty, unreacted canavanine was removed by treat- 0.999 were discarded. The turnover number was calculated ment with excess Jack bean leaf arginase. from Vmax data obtained on these plots and were based upon CH (7.5 milliunits) was reacted with 25 mM L-canavanine a molecular mass of 285 kDa. (pH 7.4) and 80 mM sodium tricine buffer (pH 7.4) containing Synthesis of Radiolabeled Hydroxyguanidine. Hydrogen 30% (vol͞vol) glycerol in a final volume of 0.5 ml for 120 min. cyanamide (0.45 mmol) was added to 0.55 mmol hydroxyl- The reaction was terminated by placing the reaction mixture in amine hydrochloride in 1.6 ml of deionized water and the pH a boiling water bath for 3 min. The turbid reaction mixture was taken to 9.3 with NaOH. A total of 100 MBq barium [14C]cy- ␮ treated with 100 l purified Jack bean leaf arginase (see below) anamide (2.15 GBq͞mmol) was treated with a 2-fold excess of for at least 7 h, but often overnight at 37ЊC.AunitofCHis sulfuric acid in 0.4 ml of deionized water; barium sulfate was that amount of enzyme that catalyzed the formation of 1 ␮mol Ϫ1 removed by centrifugation. The radiolabeled cyanamide was hydroxyguanidine͞min under the described assay condi- added to the reaction mixture and the pH re-adjusted to tions. 8.9–9.1 with dilute NaOH. [14C]Hydroxyguanidine formation The hydroxyguanidine-PCAF chromogen was produced by was allowed to continue for 48 h at 3ЊC. The radioactive adding 1.0 ml of 200 mM potassium phosphate buffer (pH 7.0) reaction mixture was taken to pH 3.5 with 2 M HCl and placed to the arginase-treated CH reaction mixture followed by 0.2 ml ϩ ona17ϫ60 mm column of Dowex 50 (NH4 ). After washing of 1.0% (wt͞vol) potassium persulfate and 0.2 ml of 1.0% the column exhaustively with deionized water, radiolabeled (wt͞vol) PCAF as described (18). The turbid colorimetric hydroxyguanidine was obtained with 300 mM ammonia. The assay tube was clarified by centrifugation with an International first 25 ml of effluent, containing radiolabeled hydroxyguani- Equipment (Needham, MA) table-top centrifuge for 1 min. At dine, was discarded to remove any unreacted cyanamide. The least 3 min but not more than 5 min after the addition of PCAF, subsequent 10 ml of effluent (fraction 2) was dried to a residue the hydroxyguanidine–PCAF chromogen was read at 475 nm. by rotary evaporation in vacuo at a temperature not more than Purified Arginase. Jack bean leaf arginase, purified through 23ЊC. Before drying, a sample of fraction 2 was used to the ammonium sulfate precipitation step and dialysis, was determine the hydroxyguanidine content by colorimetric anal- prepared as described and stored at Ϫ80ЊC (13, 19). The ysis. Sufficient carrier hydroxyguanidine, taken to pH 7.4, and thawed protein preparation, obtained from 120 g of 12-day old deionized water were added to the residue to prepare 2 ml of jack bean leaves, was placed on a 90 ϫ 300 mm column of 175 mM [14C]hydroxyguanidine that was stored at Ϫ20ЊC DEAE-cellulose equilibrated with 25 mM sodium tricine (pH overnight before use. The specific activity of the [14C]hydrox- 7.6) containing 0.1% (vol͞vol) 2-mercaptoethanol and 0.5 mM yguanidine solution was 13.2 KBq͞␮molϪ1. dithiothreitol (standard buffer). The column was washed with Polyacrylamide Gel Electrophoresis. SDS͞PAGE was con- 200 ml of 50 mM standard buffer. Arginase was obtained by ducted under denaturing conditions and 15% polyacrylamide gradient elution employing 300 ml of 50 mM standard buffer gel. Nondenaturing gels consisted of a 5–20% gradient of and an equal volume of standard buffer with 250 mM NaCl. polyacrylamide. The running buffer was 100 mM Tris͞glycine The effluent was collected as 7 ml fractions in test tubes (pH 8.3). Proteins were stained with 0.1% (wt͞vol) Coomassie containing 7 ␮mol MnCl2; the flow rate was 105 ml͞h. blue R-250. Before electrophoresis, CH was treated with SDS Downloaded by guest on September 29, 2021 Biochemistry: Michelangeli et al. Proc. Natl. Acad. Sci. USA 94 (1997) 2257

in the presence of 2-mercaptoethanol for 3 min at 100ЊC. its corresponding lactone. To verify the identity of homoserine Bio-Rad high and low SDS͞PAGE molecular weight standards by way of its lactone, the deproteinized reaction mixture, after and Sigma electrophoresis kit standards were used to deter- treatment with a vast excess of Jack bean arginase, was placed ϩ mine CH mass. ona10ϫ25 mm column of Dowex 50 (NH4 ). Homoserine was Sequence Data. Protein alkylation, enzymatic cleavage, pep- eluted with deionized water while any remaining canavanine tide purification, and peptide sequence analysis were per- was bound to the resin. The column effluent was refluxed with formed at the Macromolecular Structure Analysis Facility of 19% (wt͞vol) anhydrous HCl for 90 min. After removing the the University of Kentucky. CH (Ϸ0.1 mg) was desalted by solvent by rotary evaporation in vacuo, the residue was allowed adsorbing the protein onto a polyvinylidene fluoride mem- to dry in vacuo at 55ЊC for an additional 30 min after solvent brane held in a ProSorb Apparatus (Perkin–Elmer). The removal. Deionized water was added to the residue and the polyvinylidene fluoride-bound protein was reduced by placing drying process repeated twice. The residue was then dissolved the membrane in 90 ␮l of 100 mM Tris⅐HCl buffer (pH 8.5) in 3 M HCl and refluxed for 90 min at 115ЊC. Afterward, the with 10 ␮l acetonitrile containing 7 mM dithiothreitol at 37ЊC HCl was removed by exhaustive rotary evaporation in vacuo for 1 h, and then alkylated with 20 mM iodoacetamide in the and the residue dissolved in deionized water, taken to pH 3.5 dark at 23ЊC for 15 min. After alkylation, the membrane-bound with1MNH3, and placed on a 10 ϫ 25 mm column of Dowex ϩ protein was washed with 1 ml of deionized water, 1 ml of 100 50 (NH4 ). The column was washed with 0.7 liter of deionized ␮ mM Tris⅐HCl (pH 8.5), and then treated with 10 l acetonitrile water and developed with 0.5 liter of 200 mM NH3; the effluent plus 10 ␮l 10% hydrogenated Triton X-100 with 100 ng was concentrated by rotary evaporation in vacuo. endoproteinase Lys-C (Boehringer Mannheim) in 100 mM The above procedure converts homoserine stoichiometri- Tris⅐HCl (pH 8.5) in a final volume of 100 ␮lfor18hat37ЊC. cally to homoserine lactone; unlike homoserine, the latter The supernatant solution was saved and the membrane compound is basic. Free homoserine and any neutral or acidic washed with 100 ␮l 40% (vol͞vol) aqueous acetonitrile and components of the reaction mixture cannot bind to this resin then with the same solvent containing 0.05% (vol͞vol) triflu- in the ammonia form. Homoserine lactone is converted to oroacetic acid (TFA). The peptides of the supernatant solution homoserine in situ when the column is developed with 200 mM and pooled washes were isolated by reverse-phase HPLC using ammonia. Thus, this procedure provides a means of demon- a Hewlett–Packard model 1050 equipped with a Vydac (Hes- strating unequivocally the production of homoserine by CH peria, CA) 2.1 ϫ 250 mm C18 TP silica column. Peptides were through its lactone. Ammonia was identified as a reaction obtained by linear gradient elution with 0.06% (vol͞vol) product by automated amino acid analysis as described above. aqueous TFA and 70% (vol͞vol) aqueous acetonitrile con- Purification of Canavanine Hydrolase. Preparation of the taining 0.054% (vol͞vol) TFA over 40 min. The peaks were homogenate. H. virescens fifth instar larvae (n ϭ 125) fresh detected at 214 nm and the peptide fractions collected by hand. weight, 300–360 mg͞larva, were submerged under crushed ice All peptide sequence analysis was performed on an Applied for at least 15 min. The chilled larvae were opened with a small Biosystems model 477A peptide sequencer using Polybrene- scissor, the midgut removed from the body cavity, and the gut coated glass fiber discs and standard chromatography for contents flushed out by forcing 50 mM sodium tricine buffer phenylthiohydantoin-amino acid analysis. (pH 7.4) containing 30% (vol vol) glycerol (standard buffer) Evaluation of the Reversibility of the Reaction. Canavanine ͞ through the gut with a fine-needle syringe. The clean midguts hydrolase (160 milliunits) was mechanically agitated with 25 were homogenized with 4 ml of the above buffer; the homog- mM hydroxyguanidine (13.2 KBq͞␮molϪ1) and 25 mM L- homoserine in standard buffer containing 0.15 ml of Jack bean enizer was rinsed with 1–2 ml of the standard buffer. After leaf arginase supplemented with 2 mg jack bean seed urease clarifying the turbid solution by centrifugation at 12,000 ϫ g for 15 min, the pellet was reextracted with 2–3 ml of standard (Sigma; type III, 264 units͞mgϪ1) in a final volume of 0.7 ml at 37ЊC overnight. The reaction was initiated by injecting 0.3 buffer and processed as above. The combined supernatant ml of CH into the reaction mixture and terminated by a similar solutions were poured over cheesecloth to remove fat and administration of 1.0 ml of 2 M HCl. The acidified reaction other floating debris. mixture was shaken mechanically overnight to collect the Ammonium sulfate fractionation. The crude homogenate was evolved radioactive carbon dioxide. All assays were conducted taken to 52% (vol͞vol) saturation with liquid saturated am- in triplicate. This radiometric assay would convert any radio- monium sulfate (pH 7.2) and allowed to sit at 6ЊC for 60 min labeled canavanine, formed in the reverse direction, to radio- before centrifugation at 12,000 ϫ g for 15 min. The pellet was 14 discarded and the supernatant solution taken to 65% (vol vol) labeled urea and then CO2. The latter is trapped in hydroxide ͞ of hyamine and quantified by liquid scintillation spectroscopy; saturation and allowed to sit on ice for 90 min before centrif- this radiometric assay has been described (22). ugation. Reaction Product Identification. Hydroxyguanidine was es- CH was stored routinely at this stage at Ϫ80ЊC as a pellet tablished as a reaction product after isolating it by ionic- under frozen liquid ammonium sulfate without loss in catalytic ϩ exchange chromatography using Dowex 50 (NH4 ) after all activity. Each pellet contained 3.8 mg protein obtained from detectable canavanine had been removed by enzymatic deg- 25 larval guts. radation. The isolated hydroxyguanidine gave an absorption Acetone fractionation. Two thawed CH pellets (50 larval spectrum, over the range of 400–600 nm, that was identical to guts) were centrifuged at 11,500 ϫ g for 6 min before dissolving authentic material and different from that of canavanine. In the pellets in 5.0 ml of 50 mM tricine (pH 7.4) containing 30% addition, the isolated hydroxyguanidine was stoichiometrically (vol͞vol) glycerol. The protein was stirred mechanically in an hydrogenated to guanidine using a palladium black catalyst in ice bath and treated by the drop-wise addition of 4.0 ml (44.4% the presence of H2. Guanidine was isolated by ionic-exchange saturation) of freshly distilled acetone kept at Ϫ80ЊC imme- ϩ chromatography with Dowex 50 (NH4 ). After washing the diately before use. After standing for exactly 7.0 min at Ϫ20ЊC, resin with 3 M ammonia, a solution able to elute all hydrox- the turbid solution was clarified by centrifugation at 14,500 ϫ yguanidine and substances as basic as arginine, guanidine was g for 4 min at 1ЊC. The supernatant solution was treated with obtained by developing the column with 5 M ammonia. The 2.2 ml of acetone (55.4% saturation) and processed as de- eluted guanidine was identified by colorimetric analysis using scribed above. The pellet was dissolved in 1.2 ml of 50 mM the diacetyl reaction and guanidine⅐HCl as a standard (23). tricine (pH 7.4) containing 30% (vol͞vol) glycerol, commi- Homoserine was established as a reaction product by auto- nuted with a glass homogenizer, and centrifuged at 14,500 ϫ mated amino acid analysis of the reaction mixture. In addition, g for 12 min. The supernatant solution was purified further by the reaction mixture was processed to convert homoserine to G-200 Sephadex chromatography. Downloaded by guest on September 29, 2021 2258 Biochemistry: Michelangeli et al. Proc. Natl. Acad. Sci. USA 94 (1997)

FIG. 2. Apparent Km determination for CH. The Km value for CH FIG. 1. Elution profile from G-200 Sephadex chromatography. The was determined under standard assay conditions with 0.267 mg CH effluent protein was measured at 280 nm. CH eluted as the initial peak. purified through the G-200 Sephadex step.

1 G-200 Sephadex chromatography. Canavanine hydrolase (1.0 activity at 3ЊC when standing in buffer alone (t ͞2 Ϸ 8 h). ml), purified through acetone fractionation, was applied to a Dialysis of CH, against glycerol-supplemented buffer, caused

1 1.4 ϫ 83 cm column of G-200 Sephadex equilibrated with 50 a complete loss in catalytic activity (t ͞2 Ϸ 5 h). This loss also mM tricine (pH 7.4) containing 30% (vol͞vol) glycerol. Frac- occurred when CH was processed by ultrafiltration with an tions (1.5 ml) were collected every 15 min (Fig. 1). The two Amicon P-10 membrane. The marked lability of CH undoubt- most active fractions, Ϸ0.3 mg, were pooled and evaluated by edly resulted from a dissociation of the native enzyme which HPLC (Table 1). can be prevented with glycerol. HPLC. Trace impurities of CH, obtained by G-200 chroma- Microsome Purification. To determine if CH was a soluble tography, were removed from CH by injecting 250 ␮lofthe protein rather than one associated with the microsomal frac- pooled sample into a C18-WP column (Bakerbound, Beck- tion, a freshly prepared gut extract was centrifuged at 12,000 ϫ man); the protein was eluted with a linear gradient of 0.1% g for 15 min, and the supernatant solution was recentrifuged (vol͞vol) aqueous TFA and 80% (vol͞vol) aqueous acetoni- at 105,000 ϫ g for 60 min. Assay of the resulting supernatant trile containing 0.02% (vol͞vol) TFA over 40 min. The efflu- solution and microsomal pellet revealed that all the detectable ent was monitored at 280 nm with an Scientific Systems (State enzymic activity resided solely in the supernatant solution. College, PA) model 500 variable wavelength spectrophotom- Characterization of the CH-Mediated Reaction. The ability eter. The digitalized data were stored and processed by a of CH to cleave hydrolytically the O–N bond of the guanidi- Dionex advanced computer interface. Homogeneous CH was nooxy moiety of L-canavanine to generate L-homoserine and collected by hand employing analog output detection obtained hydroxyguanidine with the expected stoichiometric yields was with a Spectra-Physics SP-4270 automated area integrator. established by direct isolation, identification, and then quan- Criteria of purity. The purity of the H. virescens CH was titation of the reaction products by the procedures described established by PAGE at acid, neutral, and basic pH. CH purity in full above. These procedures demonstrated that reaction of was confirmed by HPLC analysis which revealed a single peak 10.0 ␮mol of L-canavanine with CH (200 milliunits), overnight using a variety of elution profiles. under standard assay conditions, yielded 9.62 Ϯ 0.4 ␮mol L-homoserine and 9.71 Ϯ 0.3 ␮mol hydroxyguanidine. These RESULTS values are within 3–4% of the expected stoichiometric yields. Kinetic Analysis. Kinetic analysis of CH revealed an appar- Electrophoretic Analysis. PAGE procedures revealed that ent Km value of 1.1 mM for L-canavanine (Fig. 2). The highest under reduced, denaturating conditions, CH ran as two bands specific activity CH gave a turnover number of 21.1 Ϯ 0.2 possessing molecular masses of 50 or 47.5 kDa. Under non- ␮mol⅐minϪ1⅐␮molϪ1. denaturing conditions, CH exhibited a mass of 285 kDa. These Substrate Specificity. We determined whether CH could data suggest that CH is a hexameric protein composed of two attack the O–N bond of the aminooxy moiety of canaline, different subunits. presumably to yield homoserine and hydroxylamine. While CH CH Stability. CH was stable for as long as 10 days at 3ЊCin degraded canaline [L-2-amino-4-(aminooxy)butyric acid], as the presence of 50 mM tricine buffer (pH 7.4) supplemented determined by colorimetric analysis of the reaction mixture, with 30% (vol͞vol) glycerol, but underwent a rapid loss in canaline loss was 4.5 ϫ 10Ϫ3 less than that observed for

Table 1. Purification of CH from H. virescens Total activity, Specific activity, Fraction milliunits Protein, mg milliunits͞mg Recovery, % Larval gut extract 690 187 3.7 Ammonium sulfate precipitation (52–65%) 505 19 26.6 73 Acetone fractionation (44.5–55.4%) 385 4.8 80.2 56 G-200 Sephadex chromatography 115 0.8 143.7 17 Experimental details are provided in the text. Downloaded by guest on September 29, 2021 Biochemistry: Michelangeli et al. Proc. Natl. Acad. Sci. USA 94 (1997) 2259

comparably processed canavanine. We also assessed the ability dominately on canavanine-free plants (26, 27), but many of CH to attack O-ureido-L-homoserine [L-2-amino-4- leguminous plants, particularly members of the Fabaceae—the (ureidooxy)butyric acid; UHS] to yield homoserine and hy- group of legumes most noted for their ability to store cana- droxyurea. UHS was such a poor substrate that compound loss vanine (28, 29)—are consumed by the larvae. It is not know was barely detectable; UHS decomposition was at least 10Ϫ3 how this insect evolved this detoxification enzyme; perhaps, less than that of canavanine. This finding undoubtedly reflects this evolved when the larvae relied more heavily on canava- steric hindrance of the bulky ureidooxy moiety of UHS in nine-containing plants as a food resource. failing to gain access to the active site of the enzyme. Given that the gut was cleansed thoroughly before extrac- Recent development of chemical methods for the synthesis tion, CH is probably an enzyme of the gut wall rather than a of the lower and higher homolog of L-canavanine, 2-amino- protein secreted into the gut cavity. The stabilizing effect of 3-(guanidinooxy)propionate, and 2-amino-5-(guanidinooxy)- glycerol on this otherwise highly labile protein undoubtedly hexanoate (homocanavanine), respectively, permitted an as- results from its ability to mimic conditions existing within the sessment of the effect of aliphatic chain length on CH activity. gut wall matrix that stabilize and support CH. Neither canavanine derivative supported detectable hydrox- Finally, prior studies of canavanine disposition in the he- yguanidine formation. In a similar vein, neither methyl, ethyl, molymph of H. virescens larvae established that an injected or butyl ester of L-canavanine yielded hydroxyguanidine on dose of 5 g L-canavanine͞kgϪ1 fresh body weight is cleared 1 treatment with CH. Our structure–activity studies established with a t ͞2 of 135 min by active metabolic processes (7). This the rigid requirement for canavanine’s unique structural fea- route of administration bypassed the gut initially and such tures for a compound to serve as an active substrate for CH. canavanine clearance from the hemolymph might be achieved Reversibility of the Reaction. The reversibility of the CH- by a CH of the fat body. mediated cleavage of L-canavanine to L-homoserine and hy- The fat body, in simple terms, is the metabolic equivalent of droxyguanidine was evaluated with a highly sensitive radio- the vertebrate liver. It is the site of synthesis of complex metric assay described above. This radiometric assay revealed carbohydrates, lipids, nonessential amino acids, and a host of that, even under conditions in which arginase pulled the structural and enzymic proteins (30). Analysis of the CH reaction in the reverse direction, only 1.2% of the [14C]hy- activity of the fat body revealed that this organ does not have droxyguanidine was converted to L-[guanidinooxy-14C]canava- any significant CH activity. While movement of CH from the nine overnight at 37ЊC. Thus, CH mediated essentially an lumen of the digestive tract into the hemolymph has not been irreversible reaction. established, enzymes can move unchanged across the gut wall Prior analysis of hydroxyguanidine metabolism in H. vire- into the hemolymph (30). Such transport may account for the scens demonstrated that this nitrogen-rich compound is re- ability of H. virescens to efficiently clear the hemolymph of duced to guanidine which is the end product of canavanine canavanine. catabolism (10). Guanidine evidently has no discernible con- sequence on larval growth and development. We gratefully acknowledge National Science Foundation Grant Primary Structural Analysis. To further characterize this IBN-9302875 and support provided by Consejo de Desarrollo Cien- larval enzyme, portions of its primary structure were deter- tifico y Humanistico and Fundacion Polar, Maracay, Venezuela. This mined. This analysis revealed structural information for three is paper 96–08-035 of the Kentucky Agricultural Experimental Sta- peptidal fragments: peptide 1, KFVPISCPMPTNRQP; pep- tion, Lexington. tide 2, KSLVVNINENSVK; and peptide 3, KMPWFTNXX- 1. Dahlman, D. L. & Rosenthal, G. A. (1975) Comp. Biochem. TYFGXT. Comparison of these sequences with primary struc- Physiol. A 51, 33–36. tural information stored in the nonredundant protein database 2. Rosenthal, G. A. (1977) Q. Rev. Biol. 52, 155–178. at GenBank, the Saccharomyces genome database, and the 3. Rosenthal, G. A. (1991) in Nonprotein Amino Acids as Protective Haemophilus influenzae genome database failed to disclose Allelochemicals, eds. Rosenthal, G. A. & Berenbaum, M. (Aca- significant structural homology with a known macromolecule. demic, San Diego), pp. 1–11. 4. Bleiler, J., Rosenthal, G. A. & Janzen, D. H. (1988) Ecology 69, 427–433. DISCUSSION 5. Rosenthal, G. A. (1983) Sci. Am. 249, 164–171. H. virescens larvae are able to tolerate extraordinarily high 6. Rosenthal, G. A., Hughes, C. G. & Janzen, D. H. (1982) Science 217, 353–355. levels of dietary canavanine even though this nonprotein 7. Berge, M. A., Rosenthal, G. A. & Dahlman, D. L. (1986) Pestic. amino acid is highly insecticidal to nonadapted animals. This Biochem. Physiol. 25, 319–326. ability results primarily from a gut enzyme, canavanine hydro- 8. Berge, M. & Rosenthal, G. A. (1990) J. Food Agric. Chem. 38, lase, that mediates an irreversible hydrolysis of L-canavanine to 2061–2065. L-homoserine and hydroxyguanidine. This detoxification 9. Berge, M. A. & Rosenthal, G. A. (1991) Chem. Res. Toxicol. 4, mechanism stands in contrast to canavanine catabolism by 237–240. larvae of the bruchid beetle, C. brasiliensis, who develop within 10. Rosenthal, G. A. (1992) Bioorg. Chem. 20, 55–61. and are nurtured by the canavanine-laden seeds of the legume, 11. Kalyankar, G. D., Ikawa, M. & Snell, E. E. (1958) J. Biol. Chem. Dioclea megacarpa (4, 5). This neotropical insect hydrolytically 233, 1175–1178. 12. Webb, E. C. (1992) Enzyme Nomenclature (Academic, San Di- cleaves L-canavanine to L-canaline and urea (5). At the same ego). time, these larvae have extraordinarily high levels of urease 13. Bass, M., Harper, L., Rosenthal, G. A., NaPhuket, S. & Crooks, (24) which enables the developing larvae to generate ammo- P. (1995) Biochem. Syst. Ecol. 23, 717–721. niacal nitrogen for the synthesis of a number of amino acids 14. Rosenthal, G. A. (1973) Anal. Biochem. 56, 435–439. (6). 15. Rosenthal, G. A., Dahlman, D. L., Crooks, P. A., NaPhuket, S. & The gut of H. virescens also contains an active L-arginine Trifonov, L. S. (1995) J. Food Agric. Chem. 43, 2728–2734. kinase (adenosine 5Ј-triphosphate: L-arginine phosphotrans- 16. Ozinskas, A. J. & Rosenthal, G. A. (1986) Bioorg. Chem. 14, ferase, EC 3.5.3.1) which catalyzes an ATP-dependent phos- 157–162. 17. Berger, R. S. (1963) USDA Res. Serv. 33, 84. phorylation of L-canavanine to yield the novel phosphagen, 18. Rosenthal, G. A. (1977) Anal. Biochem. 77, 147–151. L-canavanine phosphate (25). These two enzymes account for 19. Kavanaugh, D., Berge, M. & Rosenthal, G. A. (1990) Plant virtually all of the canavanine catabolism fostered by an extract Physiol. 94, 67–70. of the larval gut. 20. Chinard, F. P. (1952) J. Biol. Chem. 199, 91-94. H. virescens larvae are generalist feeders—i.e., they consume 21. Rosenthal, G. A. & Dahlman, D. L. (1991) J. Biol. Chem. 266, a diversified array of plants. Presently, this insect feeds pre- 15684–15687. Downloaded by guest on September 29, 2021 2260 Biochemistry: Michelangeli et al. Proc. Natl. Acad. Sci. USA 94 (1997)

22. Rosenthal, G. A. & Thomas, D. (1985) Anal. Biochem. 147, 428–431. 26. Bell, E. A., Lackey, J. A. & Polhill, R. M. (1978) Biochem. Syst. 23. Bonas, J. E., Cohen, B. D. & Natelson, S. (1978) Microchem. J. 7, Ecol. 6, 201–212. 63–77. 27. Natelson, S. (1985) J. Agric. Food Chem. 33, 413–419. 24. Rosenthal, G. A. (1990) in Bruchids and Legumes: Economics, 28. Pearson, O. (1958) The Insect Pests of Cotton in Tropical Africa Ecology and Coevolution, eds. Fujii, K., Gatehouse, A. M. R., (Commonwealth Inst. Entomol., London). Johnson, C. D., Mitchell, R. & Yoshida, T. (Kluwer, Amster- 29. Zulucki, M. P., Daglish, G., Firempong, S. & Twine, P. (1986) dam), pp. 161–170. Aust. J. Zool. 34, 779–814. 25. Gindling, H. L., Rosenthal, G. A. & Dahlman, D. L. (1995) Insect 30. Friedman, S. (1985) in Fundamentals of Insect Physiology, ed. Biochem. Mol. Biol. 25, 933–938. Blum, M. S. (Wiley, New York), pp. 467–506. Downloaded by guest on September 29, 2021