578 BIOCHIMICA ET BIOPHYSICA ACTA

BBA 26 194

BIOSYNTHESIS OF ASPARAGINE FROM fl-L-CI~'~N]CYANOALANINE IN

L A T H Y R U S S Y L V E S T R I S W. SEEDLINGS

ORIGIN OF THE AMIDE NITROGEN

CHARLOTTE RESSLER, G. R. NAGARAJAN AND CHRISTINE LAUINGER Division of Protein Chemistry, Institute for Muscle Disease, Inc. and Department of Biochemistry, Cornell University Medical College, New York, N.Y. xoo2x ( U.S.A .)

(Received March 27th, 1969)

SUMMARY

fl-L-[14C15N~Cyanoalanine administered to Lathyrus sylvestris W. seedlings form- ed asparagine with complete retention of cyano nitrogen. The fl-cyanoalanine path- way for the young plant may be a means of conserving nitrogen as well as of detoxi- fying cyanide. The required fl-t-[Cl~N~cyanoalanine was synthesized by hydrogenoly- sis of carbobenzoxy-L-fl-[Cl~N~cyanoalanine benzyl ester, prepared by dehydration of known carbobenzoxy-L-[amide-lSNlasparagine benzyl ester. (1~NH4)2S04 was the source of laN.

INTRODUCTION

In an earlier study in this laboratory fl-cyanoalanine, in the form of 7-glutamyl- fl-cyanoalanine, was established as the major product of fixation of inorganic cyanide in Vicia sativa seedlings 1, 2. In Lathyrus sylvestris seedlings, in which asparagine is the chief product of cyanide fixation, fl-cyanoalanine was established as an intermediatel, 2. The fl-cyanoalanine pathway is also present in Chromobacterium violaceum 3 and prob- ably in a variety of plants and other systems capable of incorporating administered inorganic cyanide into fl-cyanoalanine or asparaginO.

In V. sativa, CN- was incorporated as the cyano group of fl-cyanoalanine with the carbon-nitrogen bond intact 2. In L. sylvestris, the cyano carbon of fl-cyanoalanine appeared as the amide carbon of asparagine, but the fate of the cyano nitrogen was not clear. Aspartic acid was known to be formed from fl-cyanoalanine by the action of E. coli 5 and guinea pig serum asparaginases 6 and certain bacterial nitrilases ~, and, al- though less efficient than fl-cyanoalanine, it likewise served as a significant precursor of asparagine in L. sylvestris 2. Moreover, shortly after administration of 7-glutamyl- fl-cyanoalanine- [4-C14~ to cut seedlings, tissue aspartic acid had a higher specific activi- ty than asparagine, which suggested that it perhaps coxfld mediate the conversion of fl-cyanoalanine into asparagine 2. The data did not rule out the possible existence of separate, perhaps simultaneous pathways to asparagine, i.e. direct conversion of fl- cyanoalanine into asparagine, conversion of fl-cyanoalanine into aspartic acid that is converted into asparagine, and amidation of aspartic acid derived from other sources.

Biochim. Biophys. Acta, 184 (I969) 578-582

BIOSYNTHESIS OF ASPARAGINE AMIDE 579

In the present paper these first two possibilities have been evaluated by admi- nistering to L. sylvestris seedlings fl-cyanoalanine 'doubly labeled' with 14C and 15N in the cyano group and determining the ratio of these isotopes in the amide group of the formed asparagine.

For this purpose a source of L-fl-EC15N]cyanoalanine was needed. The route se- lected, which is outlined, employs synthetic procedures and intermediates in current use as well as an inexpensive source of the isotope. 15N of 96.2 a tom % enrichment was introduced as 15NHs into the amide group of carbobenzoxy-L-[amide-i~N~ aspara- gine benzyl ester (I), a known intermediate for L-Iamide-l~N]aspara6ne ~. Dehydration with dimethylformamido chloride s converted I in 58 % yield into carbobenzoxy-L- fl-ICi~N]cyanoalanine benzyl ester (II), which on hydrogenolysis afforded the desired amino acid in 36 To yield after purification.

COOCTH 7 COOC~H~ COOH

I socl, [ r,a/H, h Z N H C H Z N H C H > CHNHz [ Dimethylformamide ) [ CHsOH ]

CH~ CH~ CH z I ] I

COISNH, C ~XSN C-=15N

I II I I I Z = CeHsCH~OCO-

MATERIALS AND METHODS

Materials Source of seeds 2 and synthesis of L-fl-E14CN]cyanoalanine9 are described else-

where. (I~NH,)2S0,, 96.2 a tom %, was purchased from Isomet Corporation, Palisades Park, N. J. Solvents in synthetic work were dried over Linde 4 A molecular sieves. Melting points are uncorrected.

Synthesis of carbobenzoxy-L-fl-[O~N]cyanoalanine benzyl ester (II) Carbobenzoxy-L-[amide-lS'N]asparagme benzyl ester (I) was prepared from

carbobenzoxy-~-benzyl-L-aspartate and (15NH4)~SOt (ref. 7). Unreacted I~NH3 was recovered by Kjeldahl distillation of the aqueous filtrate of the product and reused for another preparation of I.

A solution of I (0.535 g, 1.5 mmoles) in dimethylformamide (I ml) in a T/s 19/38 test tube was maintained in an anhydrous condition and cooled in an ice bath. Thionyl chloride (0.325 ml, 4.5 mmoles) was added with magnetic stirring. After IO rain, the solution was brought to room temperature and stirred further for 14 ° min. The red solution was concentrated first at the water aspirator to remove thionyl chloride and then at o.I m m pressure t~ remove solvent. The syrup was treated with IO ml of ice- cold water. After 20 min, the resultant yellow precipitate was collected on the filter, washed with cold water, and dried over P206; wt. 480 mg, m.p. lOO-lO4.5 °. A second run with 497 mg of I I yielded 430 mg of a similar melting point. The combined crude product, 9oo mg, was crystallized from ethyl acetate-l ight petroleum (b.p. 30-60 °) and gave 560 mg (58. 4 %) of colorless leaflets, m.p. lO5-1o7% sintering at 99 °. A sec- ond crop of 9 ° mg melted at lO4-1o6 ° and a third crop of i i o mg melted at lO2-1o4 °. For analysis the material was recrystallized further from the same solvent; m.p.

Biochim. Biophys. Acta, 184 (1969) 578-582

580 C. RESSLER et al.

IO5-IO7 °, sintering at 99°; Ee;~ 6 --32o (c 0.4, ethanol). Found: C, 67.4; H, 5.37; N, 8.21. C19HxsN204 requires C, 67.4; H, 5.36; N, 8.28 %. Recrystallization from car- bon tetrachloride sharpened the melting point to lO6-1o7 ° with little loss. Properties are for the nonisotopic compound.

Synthesis of I:[LEC~SN]cyanoalanine (Ill) A solution of I I (255 mg, 0.75 mmole) in 30 ml of methanol was treated with a

stream of H~ in the presence of 12o mg of palladium black catalyst. Evolution of CO s ceased after 35 min, and the reaction was continued for the next 40 min. The catalyst then was filtered off and washed with hot water, and the combined filtrate was taken to dryness in a vacuum. Crystallization from a small volume of water-dioxane gave 4 ° mg. Recrystallization afforded 35.4 mg that was 9 ° % pure on amino acid analysis and showed a small amount of 2,4-diaminobutyric acid on paper electrophoresis. After a second recrystallization, 31 mg of I I I were obtained that was homogeneous by both criteria.

In a number of runs in methanol or dimethylformamide on a microscale (6 rag, 0.8 ml solvent, 4 mg Pd for 50 min), yields of/~-cyanoalanine directly after hydrogeno- lysis without isolation were 48-59 %. Since several ninhydrin-positive impurities are present in the crude product, it may be advantageous to purify by chromatography or electrophoresis before crystallization.

Cultural conditions and administration of labeled materials Seeds were sterilized chemically ~ and were also autoclaved for 15 min. The seeds

were allowed to germinate in the dark and were cultured as described elsewherO °. 6 flasks, each containing 27 seedlings, received 5 ml of nutrient medium. 3 days later, the roots of each group of the 4o-day-old seedlings received, through a sterile Swinny filter, 1. 5 ml of an aqueous solution of 3.525 mg of L-/3-E~4ClSNlcyanoalanine. The total L-/3-~14C15Nlcyanoalanine administered, 21.15 rag, was prepared by mixing in solu- tion 16.4o mg of L-/3-EC15N]cyanoalanine, 96.2 atom % excess, and 4-75 mg of L-/3- E14CNlcyanoalanine. The plants were irradiated daily with a Sylvania Gro-lux lamp for 4 days.

Analyses Amino acids were determined on the automatic amino acid analyzer 11. Aspara-

gine was determined indirectly by hydrolysis with guinea pig asparaginase 2. Radio- activity of purified compounds was determined by liquid scintillation counting in Bray 's mixture containing 3.3 % NCS solubilizer (Nuclear Chicago). Correction was made for quenching TM. Seedlings first were converted by wet oxidation to CO s (ref. 13) which then was counted by liquid scintillation. Radiochemical purity and distribution of radioactivity in extracts were assessed by scanning the column effluent of the amino acid analyzer by liquid flow scintillation before the reaction with ninhydrin 1~. 15N was determined by mass spectrometry by Analytica Corporation, New York.

Isolation of asparagine Seedling extracts were prepared as described 2 and were electrophoresed at

pH 5.7; the neutral material so obtained was re-electrophoresed at pH 8.5. (ref. 2) Blocks (12 cm × 39 cm × o.5 cm) of Solka-Floc were used as the supporting medium

Biochim. Biophys. Acta, 184 (1969) 578-582

BIOSYNTHESIS OF ASPARAGINE AMIDE 581

at 5 ° with a voltage gradient of 8 V/cm. The fraction 4.5-14 cm from the origin towards the anode, located by its characteristic initial yellow color with ninhydrin, was eluted and contained 0.33 mmole (5o.1 mg) of asparagine hydrate. I t was concentrated to several ml, filtered, adjusted to pH 2, and applied to a column (I cm X 18 cm) of Dowex 50 (H+), lOO-2OO mesh. This was washed with 80 ml of water, then eluted with 3 M NH 3. After removal of NH a, asparagine was allowed to crystallize from hot water-ethanol ; wt. 45 rag. Two recrystallizations from water afforded 4 ° and 35.1 mg. The latter material was chemically and radiochemically homogeneous on the amino acid analyzer.

RESULTS AND DISCUSSION

The high content of asparagine allowed it to be isolated, repeatedly crystallized and degraded to aspartic acid and NH 3 without requiring dilution with carrier. In forming asparagine, fl-cyanoalanine was diluted only lO.6 times in specific activity and was 45.2 % incorporated (Table I). Approx. 3 % of the starting activity was asso- ciated (bound or possibly adsorbed) with the fraction insoluble in 30 % ethanol that contained protein. These observations confirm the efficacy of fl-cyanoalanine as a precursor of asparagine and the finding that asparagine is not metabolized rapidly in this young planO,3.

Asparagine amide had 7.o5 atom % I~N. Like the cyano carbon, the cyano ni- trogen of/3-cyanoalanine was diluted lO.6 times in forming asparagine amide: both in the isolated asparagine and the administered/~-cyanoalanine, the ratio of 15N to 14C w a s 1 .56 . IO -a (Table I). It is clear that/3-cyanoalanine is converted into asparagine with net retention of its cyano nitrogen. Any overall reaction of/3-cyanoalanine to yield aspartic acid that is reamidated to asparagine must be negligible if the latter step involves equilibration with NH~ from other sources. Likewise, any deamidation of asparagine by the action of asparaginase followed by reamidation as a separate step must be negligible at this stage of the plant's development. Since /3-cyanoalanine mediates the conversion of cyanide into asparagine in L. sylvestris seedlings 1, 2, the nitrogen of cyanide may be expected to be retained on incorporation into asparagine. Recently in S. vulgare seedlings CN- was shown to enter the amide group of asparagine in this way without separation of carbon and nitrogen ~5. Unlike V. sativa and L. syl-

TABLE I

INCORPORATION OF L-~-[IIC15N]CYANOALANINE INTO L-ASPARAGINE BY L. sylvestris W. SEEDLINGS

Specific activity 15N Total Total activity Incor- Ratio of × Io .3 (atom% amount × zo -6 porated lSN to x4C (counts/rain per I~mole) excess) (l~moles) (counts/rain) (%) × ro 3

L-/~- E14C15N]Cy an°" . alanine adminis tered 47.8 74.69 I85 8.84 1.56

L-Asparagine** isolated 4.5 7.o5 *** 878 3.97 45.2 1.56§

* Uptake was 99.4 % of adminis tered radioact ivi ty; 6.1. lO 4 counts / ra in were left in the medium. "" In dried plants, concentrat ion of asparagine was 6.8 ~o; of glutamine, 0.9 ~o.

** * Amide nitrogen. § For the specific ac t iv i ty of the amide carbon, specific act ivi ty of asparagine was used on the basis

of a previous exper iment in which less t han i °/o of asparagine act ivi ty was in carbons i -3 (see refs. I and 2).

Biochim. Biophys. Acta, 184 (1969) 578-582

582 c. RESSLER eb al.

vestris, S. vulgare does not appea r to conta in ~-cyanoalanine , and the repor ted reten- t ion of cyanide n i t rogen would be in good accord wi th the opera t ion of f l -cyanoalanine p a t h w a y for conver t ing cyanide into asparagine in sorghum, as i t is in L. sylvestris.

The ear ly l i t e ra ture cor re la ted the synthesis of asparagine in the young seedling wi th the d i sappearance of seed pro te in and the high metabo l ic ac t iv i ty of the amino acids formed the reby TM. Asparag ine was considered to be a s torage form of NH~ pro- duced b y such metabol i sm. At present , the a lpha carbon and amino ni t rogen of cer- ta in amino acids are t hough t to be a source of cyanide in some p lan t s and microorga- nisms 4. Al though i t is not ye t clear to wha t ex ten t the f l -cyanoalanine p a t h w a y con- t r ibu tes to the biosynthesis of asparagine compared to o ther possible routes, the de- m o n s t r a t e d re ten t ion of cyano ni t rogen as asparagine amide ni t rogen and the obser- va t ions on the s t ab i l i t y of asparagine amide carbon and ni t rogen are consis tent wi th the ear ly concept of asparagine as a nontoxic conservat ion p roduc t of n i t rogen for young plants .

ACKNOWLEDGMENTS

W e t h a n k Dr. M. M. Brysk for helpful suggest ions on steri le cul ture and Joan A. Mineo for assis tance wi th amino acid analyses.

This work was a ided b y Gran t NB 04316 from the U. S. Publ ic Hea l th Service and b y Muscular D y s t r o p h y Associat ions of America .

REFERENCES

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Biochim. Biophys. Acta, 184 (1969) 578-582