BIOCHIMICA ET BIOPHYSICA ACTA 71

BBA 25767

STUDIES ON T H E BIOSYNTHESIS OF fl-N-OXALYL-L-O~,fl-DIAMINO-

PROPIONIC ACID, T H E L A T H Y R U S S A T I V U S NEUROTOXIN

K. MALATHI, G. PADMANABAN, S. L. N. RAO AND P. S. SARMA Department of Biochemistry, Indian Institute of Science, Bangalore (India)

(Received November 29th, I966)

SUMMARY

The biosynthesis of fl-N-oxalyl-L-a,fl-diaminopropionic acid (ODAP), HOOC. CO. NH. CHz. CH(NH,). COOH is of interest, since this neurotoxin has been isolated from the seeds of Lathyrus sativus, the consumption of which causes the disease neuro- lathyrism in humans. The concentration of this non-protein amino acid in the seeds increases on germination. When the seeds are germinated in the presence of [14C2]- oxalic acid, the isolated ODAP is labelled exclusively in the oxalyl moiety. An oxalyl- CoA synthetase requiring the obligatory presence of ATP, CoA and Mg ~+ can be demonstrated in crude extracts of the seedlings. When L-a,fl-diaminopropionic acid is incubated with the enzyme in the presence of the components for oxalyl activation, net formation of ODAP can be shown. The enzymic reaction is specific to the fl-amino group of L-a,fl-diaminopropionic acid, and the higher homologues like a,y-diamino- butyric acid, ornithine and lysine are inactive in this system. ODAP is not formed with a,fl-diaminopropionic acid when the enzyme extract is prepared from Pisum sativum although oxalyl-CoA formation can be demonstrated.

INTRODUCTION

The human consumption of the seeds of Lathyrus sativus has been known to cause the disease neurolathyrism, a syndrome restflting in spastic paralysis of the legs 1. The isolation of fl-N-oxalyl-L-o=,fl-diaminopropionic acid (ODAP), HOOC.CO.NH. CHz. CH(NH~). COOH from the seeds of L. sativus ~,3 and the finding that this amino acid has potent neurotoxic action in different animals*, 5, indicates the possibility that this compound may be a responsible factor in the etiology of human neurolathyrism. BELL 6 in a detailed analysis of the free amino acid pool has detected ODAP and its higher homologue, y-N-oxalyl-L-a,y-diaminobutyric acid, in different species of Lathyrus. In addition, free a,y-diaminobutyric acid has been detected in some Lathyrus 7 and Vicia species. Free a,fl-diaminopropionic acid is present in Viaia baicalensis e and Mimosa 8. However, L. sativus contains only ODAP and neither its higher homologue nor free L-a,fl-diaminopropionic acid (DAP) can be detected in these seeds. In addition to these, the Lathyrus species contain unusual amino acids like homoarginine 9 and lathyrine 1°. The presence of such unusual amino acids in Lathyrus

Abbreviations: ODAP, fl-N-oxalyl-L-O~,fl-diaminopropionic acid; DAP, L-0~,fl-diamino- propionic acid.

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72 K. MALATHI et al.

has been taken to suggest that although the free non-protein amino acid nitrogen may be very much less than the protein nitrogen stored in a seed, it may nevertheless constitute a small highly concentrated reserve immediately available to the embryo on germination e. In the present studies at tempts to demonstrate a possible enzymic breakdown of ODAP under these conditions have not been successful. On the contrary evidence has been obtained for a net synthesis of this oxalyl amino acid on germination and the details of this s tudy are presented in this paper.

MATERIALS AND METHODS

Estimation of ODAP in the seeds after germination I-g lots of L. sativus seeds were germinated in petri dishes for different intervals

of time. Each lot was ground with 25 ml of 75 % ethanol along with glass powder and, after brief heating on a boiling-water ba th for 30 min, the extract was centrifuged. The supernatant was concentrated to dryness under vacuum. The dry material was taken up in a known volume of water and suitable aliquots were subjected to electro- phoresis at pH 3.6, and ODAP was estimated on the electrophoretic strip after spraying with ninhydrin and elution.

E14C21oxalic acid incorporation into ODAP in L. sativus seeds ODAP was isolated and purified from seedlings germinated in the presence of

[14C21oxalic acid as described earlier 2. Briefly, IOO g of the seeds were germinated in the presence of 0.2 mC of E14C21oxalic acid for 96 h. The seedlings were repeatedly washed with water to remove the extraneous radioactive organic acid and then ground with 75 % ethanol and glass powder in a Virtis homogeniser. The extract in a total volume of I 1 was refluxed on a boiling-water bath for 4 h and filtered. The filtrate was concentrated to about 50 ml volume in a flash evaporator at 50 °. The dark-brown colored extract was then shaken with twice the volume of chloroform and the aqueous layer was collected. The chloroform layer was washed twice with water. The aqueous layers were pooled and applied to a Dowex 50 H+ (20 cm × 2 cm) column. ODAP was eluted from the column with I 1 of water, after discarding the initial 15o ml. The eluate was concentrated and ODAP was precipitated with acetone. Repeated recrystallizations were carried out with acetone. The yield of ODAP in this experiment was 27 ° mg. Weighed amounts of the recrystallized material were used for radio- activity determinations. The specific activity of free oxalic acid present in the etha- nolic extracts of the seedlings was also determined. For this, the breakthrough fluid from the Dowex 5 ° H + column and the 15o ml of water wash were pooled and concen- t ra ted to about io ml at 5 o°. The concentrate was treated with IO ml of IO % CaC12. The precipitate formed was acidified with acetic acid and the insoluble crystalline residue was dissolved in HCl (I : i) and reprecipitated with ammonia. The acidification and ammoniacal precipitation procedures were repeated thrice. The final preparation of calcium oxalate was dissolved in I M H~SO4 and t i trated against standard KMnO 4 for estimation. Aliquots were used for planchetting and for checking the purity of the preparation by chromatography.

Distribution of radioactivity in ODAP 5-mg portions of labelled ODAP were subjected to acid hydrolysis by heating

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~-N-oxALYL-L-~,~- DIAMINOPROPION IC ACID 73

with 2 ml of 2 M HC1 at IOO ° for I h. Hydrolysis was complete under these conditions as indicated by quantitative recoveries of equimolar amounts of DAP and oxalic acid. DAP was isolated from one set of hydrolysates by preparative electrophoresis at pH 3.6 on Whatman No. 3 MM paper. The position of DAP on paper was revealed by spraying ninhydrin on a reference strip containing an authentic sample. DAP was eluted from paper with water and aliquots were used for planchetting and estimation. Oxalic acid was isolated from another set of hydrolysates by repeated ether extraction. The ether extract was evaporated to dryness and oxalic acid was isolated as calcium oxalate. This was dissolved in I M H2SO , and aliquots were used for radioactivity measurements and estimation.

Radioactivity measurements These were made in a Panax Scaler (Surrey, U.K.) using a thin-end-window

counter. Suitable corrections for background were applied. Infinitely thin samples were used for counting.

Preparation and assay of oxalyl-CoA synthetase io g of seeds were germinated for 96 h and then extracted in the cold with

0.05 M potassium phosphate buffer (pH 7.5) containing I mM GSH. The extract was centrifuged at IOOOO × g for I5 min and the contents of the endogenous ODAP and other amino acids could be greatly reduced by passing 5-ml extracts through a Sephadex column (24 cm × 2 cm) G-25 (coarse). The hydroxamate assay method was followed and was similar in details to that described by GIOVANELLI n. The enzyme was incubated with a mixture containing the following components (in t~moles) in a final volume of 3.0 ml: ATP, 20; CoA, 0.2; GSH, IO; potassium oxalate, 40; MgCI~, io; hydroxylamine, 800; potassium phosphate buffer, 200. The final pH of the incu- bation mixture was 7-5. After 2 h incubation at 37 ° the reaction was stopped by adding I.O ml of FeCI~ reagent containing 20 % (w/v) FeCls and 6.6 % (w/v) trichloro- acetic acid in 1.3 M HC1. The precipitated protein was removed by centrifugation and the supernatant color was estimated in a Klett colorimeter with 54 filter. As indicated by GIOVANELLI n the blank colour was measured with the incubation mixture to which the FeC13 reagent was added at zero time.

Formation of ODAP in cell-free extracts The enzyme was incubated in a total volume of 2.5 ml at 37 ° for 2 h with all

the components of the oxalyl-activating system together with 7.0 t~moles of D A P - HC1 at pH 7.5. The reaction was stopped with trichloroacetic acid, IO % final concen- tration. The supernatant after ether extraction was analysed by electrophoresis for the presence of ODAP.

Electrophoretic and chromatographic analyses of ODAP and its hydrolytic products The solvent systems employed have been described2, ~. ODAP and DAP were

analysed by paper electrophoresis at pH 3.6 using pyridine-acet ic ac id-water (I:iO:19o) for 2 h at 800 V. ODAP being more acidic than aspartic acid has the highest mobili ty towards the anode. DAP has the characteristic cathode mobility of a basic amino acid. ODAP could be easily separated from the ~-N-oxalyl isomer by carrying out the electrophoresis using IO % acetic acid (v/v) for 90 min at 8o0 V.

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74 i<. MALATHI et al.

The RF values of ODAP were compared with those of the enzymic product in the following solvent systems: (a) Collidine-water (12:44, ammonia atmosphere), (b) n -bu tano l -n -propano l -o . I M HC1 (I : I : I). For DAP, the solvent systems (c) iso- p ropano l -wa te r - ammon ia (4o: Io : I ) and (d) p h e n o l - e t h a n o l - w a t e r - a m m o n i a (15o:4o: IO : I) were used. For oxalic acid, (e) n-butanol-acet ic ac id-water (12 : 3 : 5) and (f) e t h a n o l - a m m o n i a - w a t e r (16:1:3) solvent systems were employed.

Estimation of ODAP and DAP on electrophoretic strips The procedure of GIRl, RADHAKRISHNAN AND VAIDYANATHAN TM was followed.

After electrophoresis of the samples, the paper strips were dried at room temperature and sprayed with a 0.5 % (w/v) solution of ninhydrin in acetone. The strips were again dried and heated at 60 ° for 30 rain. The amino acid spots were then eluted in 4 ml of 75 % ethanol containing 0.2 mg CuSO 4. After IO min the colour was measured in a Klett colorimeter with 54 filter. Appropriate standards were included in each run.

RESULTS

Table I indicates the concentration of ODAP in the seeds as a function of the germination period. I t can be seen that there was an increase in the concentration of this amino acid with a sharp rise after 72 h. Since it is a non-protein amino acid, this increase can be explained as a result of net synthesis. In addition, a t tempts to demonstrate a degiadation of ODAP to DAP and oxalic acid as well as its possible utilization by transaminatien and decaiboxylation were not successful in enzyme extracts prepared from root, shoot and cotyledons.

TABLE I

ODAP CONTENT IN THE SEEDS OF L. sativus ON GERMINATION

I-g lots were extracted wi th 75 % ethanol and ODAP was est imated on paper-eleetrophoret ic strips using n inhydr in spray and elution. Electrophoresis was carried out at p H 3.6 for 2 h at 800 V. The experimental details are given in MATERIALS AND METHODS.

Period of ODAP concn. germination (h) #moles/g seed

O 22.0 24 26.4 48 26.O 72 27.5 96 35.O

MURTI AND SESHADR113 put forward a hypothetical scheme in which ODAP can arise by oxalylation of DAP and the latter may be formed from serine or aspara- gine. In the present studies, the possible involvement of an oxalyl moiety in the synthesis of ODAP was investigated. The seeds were germinated in the presence of [14Cz]oxalic acid for 96 h. ODAP was isolated and recrystallized as described in MATERIALS AND METHODS. The data presented in Table I I indicate that the isolated ODAP was labelled exclusively in the oxalyl moiety. The specific activities of ODAP and of the oxalic acid liberated on acid hydrolysis of the parent compound were

Biochim. Biophys. Acta, 141 (I967) 71-78

f l - N - o x A L Y L - L-~,fl-DIAMINOPROPIONIC ACID 75

T A B L E II

[llC2]oXALIC ACID INCORPORATION INTO O D A P ON GERMINATION

IOO g seeds were g e r m i n a t e d wi th o.2 mC of [t4C~]oxalic acid for 96 h. O D A P was isolated and recrysta l l ized f rom t h e e thanol ic ex t r ac t s of t he seedlings. Weighed quan t i t i e s of t he c rys ta l l ine ma te r i a l were used for de t e rmin ing t he to ta l r ad ioac t iv i ty and t he d i s t r ibu t ion a m o n g t he hydro - ly t ic p roduc t s . The expe r imen ta l deta i ls are g iven in MATERIALS AND METHODS.

Specific radioactivity of the isolated compounds counts~rain per pmole

ODA P Hydrolytic products of ODA P

Oxalic acid DAP Free oxalic acid

44 ° 432 Nil 398

similar. Also, the specific activities of the oxalic acid liberated from ODAP and that of free oxalic acid in the plant were of the same order. All these results indicate that oxalic acid is incorporated into ODAP as an intact segment.

This would involve a possible activation of oxalic acid, and GIOVANELL111 has established the presence of oxalyl-CoA synthetase in Pisum sativum. The data pre- sented in Table I I I indicate that such an enzyme can also be demonstrated in L. sativus seedlings. The product formed was confirmed to be oxalyl monohydroxamate on the basis of co-chromatography with an authentic sample in solvent systems described by GIOVANELL111. The formation of oxalyl hydroxamate showed an absolute require- ment for CoA, ATP, and Mg 2+.

T A B L E I I I

ASSAY OF O X A L Y L - C o A SYNTHETASE

The comple te reac t ion m i x t u r e for oxaly l ac t iva t ion was i ncuba t ed for 2 h a n d t he oxaly l h y d r o x - a m a t e fo rmed was e s t i m a t e d as descr ibed in MATERXALS AND METHODS. 7 m g of p ro te in was inc luded in t he a s s a y mi x t u r e . S t a n d a r d p o t a s s i u m oxalyl m o n o h y d r o x a m a t e was p repa red ac- cord ing to GIOVANELLI n .

Conditions Oxalyl hydroxamate (pmoles)

Comple te 3.3 Comple te (heated enzyme) o Comple te minus A T P o Comple te minus CoA o Comple te minus Mg ~+ 0.2 Comple te minus oxa la te o. 5

The results presented in Table IV indicate that an amino acid with the same electrophoretic mobility as ODAP was formed when the enzyme was incubated with DAP and the components for oxalyl activation. Omission of CoA and ATP did not result in the formation of this amino acid. Oxalate and Mg 2+ were essential for its synthesis. In the absence of DAP there was a small but significant formation of this compound. When the complete reaction mixture was incubated there was stoichio- met ry between the amount of ODAP formed and that of DAP utilized.

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76 K. MALATHI et al.

T A B L E I V

FORMATION OF O D A P IN CELL-FREE SYSTEM

T h e c o m p l e t e r e a c t i o n m i x t u r e for o x a l y l a c t i v a t i o n w a s i n c u b a t e d a l o n g w i t h D A P for 2 h. 7 m g of p r o t e i n w a s i n c l u d e d in t h e a s s a y s y s t e m . O D A P w a s e s t i m a t e d on p a p e r a f t e r e l ec t ro - p h o r e s i s of t h e p r o d u c t a t p H 3.6 b y n i n h y d r i n s p r a y . T h e d e t a i l s a r e g i v e n in MATERIALS AND METHODS.

Conditions ODAP formed D A P utilized* (Izmoles) (l, moles)

C o m p l e t e 1.54 1.72 C o m p l e t e ( h e a t e d e n z y m e ) O . l i - - C o m p l e t e minus A T P o . i o - - C o m p l e t e minus CoA o . i i - -

C o m p l e t e minus Mg o.2o - - C o m p l e t e minus o x a l a t e o . i 2 - - C o m p l e t e minus D A P 0.39 - -

* D A P d i s a p p e a r a n c e w a s t oo i n s i g n i f i c a n t t o m a k e a n a c c u r a t e m e a s u r e m e n t in cases o t h e r t h a n c o m p l e t e s y s t e m .

T A B L E V

CHROMATOGRAPHIC MOBILITIES OF THE ENZYME PRODUCT AND ITS PRODUCTS A AND n OF ACID HYDROLYSIS

RF values

Enzyme ODA P A DA P B Oxalic acid product

0.09 (a) 0.09 (a) o.17 (c) o . I 7 (c) 0.24 (e) 0.24 (e) o.2 (b) o.2 (b) o.41 (d) o.41 (d) o.o 7 (f) 0.07 (f)

T h e l e t t e r s in p a r e n t h e s e s r e fe r to t h e s o l v e n t s y s t e m s d e s c r i b e d in MATERIALS AND METHODS.

The enzymically formed compound and its products of acid hydrolysis, A and B, co-chromatographed with authentic samples of ODAP, DAP and oxalic acid, re- spectively, in different solvent systems as can be seen from the results of Table V. DAP was further identified by its electrophoretic mobility at pH 3.6. The presence of oxalic acid was confirmed on the basis of its colour reaction with diphenylamine 14. The presence of a free a-amino group in the oxalyl amino acid formed was demon- strated by the failure to observe the characteristic ninhydrin colour after spraying with pyridoxal phosphate 1~ and copper nitrate ~6 reagents. Further, this compound had the same electrophoretic mobility as ODAP in io % acetic acid (w/v) as is depicted in Fig. I. In this system ODAP was found to be distinctly separated from the a-N- oxalyl derivative. All these properties indicate that the enzymically formed product was only ODAP. The enzymic reaction was specific to DAP since the higher homo- logues of ODAP could not be detected when, in place of DAP its higher homologues like cc,v-diaminobutyric acid, ornithine and lysine were included in the assay system.

Control experiments were performed with P. sativum which exhibits an oxalyl- CoA synthetase activity n. I t was found that despite the presence of the oxalyl-

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fl- N-OXALYL-L-cG~-DIAMINOPROPIONIC ACID 77

activating enzyme, there was no synthesis of ODAP when DAP was included in the assay system.

®

G G G C

Fig. i. Electrophoretic mobility of the enzyme product. The electrophoresis was carried out on Whatman No. I strips using lO% acetic acid (v]v) for 9o min at 8ooV. I. Enzyme product. 2. Mixture of fl-N-oxalyl-L-O',fl-diaminopropionic acid and ~-N-oxalyl-L-*¢,fl-diaminopropionic acid. 3. fl-N-oxalyl-L-O~,fl-diaminopropionic acid.

DISCUSSION

The increase in the content of ODAP on germination points to a net synthesis of this compound under these conditions. The incorporation of [14C~loxalic acid as an intact moiety into ODAP when the seeds were germinated in the presence of the labelled organic acid and the subsequent detection of the oxalyl-activating enzyme and the formation of ODAP when DAP was included in the assay system, indicate that the synthesis of the oxalyl amino acid can proceed by condensation of oxalyl-CoA with the fl-amino group of DAP. The enzyme specificity for the condensation reaction is evident on account of the preferential oxalylation of the fl-amino group, the absence of any reaction when ~,7-diaminobutyric acid, ornithine and lysine were substituted for DAP in the assay system and the lack of ODAP-synthesising activity in extracts of P. sativum, which has an oxalyl-CoA synthetase but in which seeds ODAP is not present. However, it has not been possible to detect any free DAP in the L. sativus seeds even after germination. Nevertheless, BELL s has detected ~-N-oxalyl-L-~,~ diaminobutyric acid in certain other Lathyrus species in which free ~,7-diaminobutyric acid is also present. I t is possible that in L. sativus any DAP formed is rapidly fixed as the oxalyl derivative. In this context it is of interest to note that even in the absence of added DAP there is a small but significant amount of ODAP formation in cell-free extracts (Table IV). The possible presence of an endogenous precursor for DAP which itself may be another derivative of DAP has yet to be investigated. For example N-acetyl-e~,7-diaminobutyric acid has been detected in Euphorbia pulcherima 17. Further studies are required to understand the mechanism by which the amino acid fragment of ODAP is biosynthesised in the plant.

Since ODAP does not appear to undergo degradation on germination it is possible that it may undergo breakdown under unfavourable conditions of growth. I t should be noted in this connection that L. sativus is a hardy plant growing in regions of low rainfall and needs very little agricultural operation. I t is therefore very likely that these unusual non-protein amino acids serve as a concentrated source of nitrogen under such conditions of drought rather than at the time of germination when protein breakdown can provide the necessary nitrogen reserve.

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78 K. MALATHI et al.

ACKNOWLEDGEMENT

T h e f i n a n c i a l a s s i s t a n c e o f t h e C o u n c i l o f S c i e n t i f i c a n d I n d u s t r i a l R e s e a r c h ,

N e w D e l h i , i s g r a t e f u l l y a c k n o w l e d g e d .

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