BIOSYNTHESIS OF ITACONIC ACID IN ASPERGILLUS TERREUS

BIOSYNTHESIS OF ITACONIC ACID IN ASPERGILLUS TERREUS

I. TRACER STUDIES WITH V-LABELED SUBSTRATES*

BY RONALD BENTLEY AND CLARA P. THIESSEN

(From the Department of Biochemistry and Nutrition, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania)

(Received for publication, October 15, 1956)

Large amounts of various metabolites accumulate in the culture medium of some molds grown on high sugar concentrations (1). These so called “shunt metabolites” may be produced only by molds, but seem frequently to be related to metabolic sequences well known in other organisms. In most cases, however, the nature of this relationship is obscure. A typical shunt metabolite is itaconic acid (methylene succinic acid), formed in large amounts during the growth of Aspergillus terreus NRRL 1960 on a medium of low pH (2-4). The mechanism of the biosynthesis of itaconic acid has remained unknown.

Kinoshita (5, 6) originally observed that itaconic acid was formed during growth of Aspergillus itacmicus on acid medium; when the culture medium was maintained at a higher pH by addition of calcium carbonate, calcium citrate and calcium gluconate accumulated without formation of itaconic acid. Accordingly, Kinoshita suggested that itaconic acid was formed by decarboxylation of cis-aconitic acid, the latter being derived from citric acid. In acid culture media, neither citric nor cis-aconitic acid could be detected. (Kinoshita’s strain of A. itaconicus has now largely lost the ability to form itaconic acid (7) and all subsequently described work will refer to strains of A. terreus.) Walker (8) considered that cis-aconitic acid was unlikely to be the precursor of itaconic acid; he suggested that itaconic acid was formed by condensation of 2 molecules of pyruvate, followed by dehydration and oxidative decarboxylation.

It seemed probable that a decision between these two mechanisms could be made by tracer experiments. The present paper describes a chemical degradation of itaconic acid which can be used to locate radioactivity in each carbon atom. The distribution of Cl4 in itaconic acid derived from C14-labeled glucose, acetate, and succinate has been determined.

* This work was begun by one of us (R. B.) in the Department of Biochemistry, College of Physicians and Surgeons, Columbia University, with the aid of a grant from the Nutrition Foundation, Inc. It has since been continued at the University of Pittsburgh under a grant from the National Science Foundation (NSF-G-425). Both of these grants are gratefully acknowledged. Preliminary reports of some of this work have already appeared (38).

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674 BIOSYNTHESIS OF ITACONIC ACID. I

Materials and Methods

A. terreus NRRL 1960, obtained through the courtesy of the Northern Regional Research Laboratory, was maintained on Czapek-Dox agar slants (9). Experimental cultures were grown on the corn steep-liquor- containing medium of Lockwood and Ward (2), which will be referred to as “growth medium.” Spore cultures were also prepared as described by these authors. For experiments with labeled acetic and succinic acids, cultures were first grown as surface mycelia on 100 ml. portions of growth medium in cotton-plugged 500 ml. Erlenmeyer flasks. At the appropriate time, a solution of the labeled compound was introduced below the mycelium, and the cot.ton plug replaced with a previously sterilized rubber stopper, equipped with cotton-plugged air inlet and outlet tubes and with

5 2 1 5 CH =C 2 -COOH 0-CH

! \ 2 1 H$‘, 100 52 3 4 1

/

C-CGGOH 0’ OHC.CH2.CH2.COOH + CO2

6H2 -COOH OC-CH2

3 4 4 3 Itaconic acid Aconic acid Formyl propionic

acid

Wolff - Ykishner 5 2 3 4 Successive Schmidt CH3.CH2.CH2.COOH

Butyric acid degradations ) C02from carbons

reduction 2, 3, 4, and 5. FIG. 1. Reactions used for chemical degradations of itaconic acid

a sampling tube (10). A slow stream of humidified air was passed through the flasks, CO2 in the effluent stream being absorbed in alkali scrubbers. All cultures were grown at 28-30”. Itaconic acid determinations were made by a micromodification of the method of Friedkin (11) or by the calorimetric permanganate method (12).

Labeled Xubstrates-l-C14-n-Clucose was synthesized by the method of Sowden (13). Sodium 1-C14-acetate and l-C*- and 2-C14-succinic acids were commercial preparations obtained on allocation of the United States Atomic Energy Commission. Samples for determinations of radioactivity were prepared as described by Popjak (14).

Degradation of Itaconic Acid-The degradation reactions and numbering system which will be used for itaconic acid are shown in Fig. 1.

Aconic acid was prepared by modifications of the method of Campbell and Hunt (15); although the most recent evidence suggests that the structure of aconic acid is more correctly represented as the y-lactone of p-car-


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R. BENTLEY AND C. P. THIESSEN 675

boxy-y-hydroxy-cr , p-butenoic acid (16)) it may undergo prototropic re- arrangement to the structure shown in Fig. 1 during decarboxylation. This decarboxylation was carried out by modifications of the method of Ellinger (17). Without isolation, the formylpropionic acid was reduced to butyric acid (18); the presence of formylpropionic acid was confirmed by preparation of its p-nitrophenylhydrazone from a small sample of the solution. Successive Schmidt degradations, starting with butyric acid, were carried out exactly as described by Phares (19).

Results

Fermentation of 1 -C’4-o-Glucose-A typical fermentation will be described in detail to illustrate the steps involved in all of these experiments.

TABLE I

Carbon Dioxide Production during Fermentation of I-C14-Glucose

Day No. Weight of BaCOa Specific radioactivity Total radioactivity per 24 111s.

m. PC. fier mole 10-a PG.

0.916 1.48 6.9 1.031 8.50 44.5 1.758 11.93 106.7 3.426 10.07 170.7 3.037 7.75 116.7 2.131 10.65 115.3 3.153 12.98 207.8

I I I

COz produced over 24 hour periods during the fermentation was absorbed in NaOH scrubbers and converted to barium carbonate. Total recovery of Cl4 in CO2 = 0.77~~. (16.6 per cent of that added as glucose).

l-C14-n-Clucose (6.36 gm., 140 PC. per mole = 4.64 PC.) was used to pre- pare 40 ml. of growth medium. The solution in a 200 ml. Erlenmeyer flask, equipped with air inlet and outlet tubes and a sampling tube, was sterilized and inoculated. Barium carbonate samples were prepared from daily collections of COZ. The yields and specific radioactivities of these samples are shown in Table I. Fermentation was stopped on the 8th day, since itaconic acid analysis then indicated a high yield (51 mg. per ml.). The mycelium had a dry weight of 702.9 mg. and a specific radioactivity of 0.84 X 10M3 ,UC. per mg. Total activity recovered in mycelium, 0.59 PC., i.e. 12.7 per cent of that originally added.

The culture medium was evaporated to dryness in vacua and the residue was extracted first with hot ethanol (2 X 30 ml.), and finally with hot n-butanol (2 X 30 ml.). Evaporation of these combined cxtracts and vacuum sublimation of the residues (bath temperature, 120’) on to a cold


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finger condenser gave a total of 1.76 gm. of itaconic acid, m.p. 159-160”; specific radioactivity, 102 PC. per mole. The total recovery of Cl4 in itaconic acid was 1.38 PC. (29.8 per cent of that added as glucose). As a check on the purity of the itaconic acid, a small sample was converted to l-phenyl-2-pyrrolidinone-4-carboxylic acid by brief heating with a slight excess of aniline (20). The derivative, recrystallized from water, had a melting point of 189” and a specific radioactivity of 100.5 PC. per mole.

Preparation of Sodium Aconate-The above itaconic acid (705 mg.) was suspended in water (0.9 ml.) and treated with bromine (0.3 ml.) in a small stoppered vial. The bromine was added all at once, with vigorous shak- ing; cooling was not necessary. After 5 minutes, most of the bromine had reacted. The vial was then allowed to stand unstoppered until only a trace of bromine remained (about 15 minutes). The solution, neutralized by cautious addition of sodium bicarbonate (915 mg.), was heated to 50”, and sodium carbonate monohydrate (340 mg.), dissolved in a minimal volume of water (0.6 ml.), was added dropwise with stirring, the solution being held at 50” for 10 minutes. A further small amount of sodium carbonate was then added until the solution reached pH 7.0. Separation of sodium aconate took place quickly and was completed by keeping the mixture at 0” overnight. The thick, pasty mass was filtered, washed with a little ice-cold 80 per cent alcohol, and finally with pure alcohol. The yield of air-dried sodium aconate (trihydrate) was 610 mg.

Preparation of Aconic Acid-Sodium aconate trihydrate (610 mg.) in water (10 ml.) was passed through a column of Amberlite IR-120 (H), 15 X 1.5 cm. diameter; the column was washed through with water, a total of 250 ml. of effluent being collected. Water was removed by vacuum evaporation (bath temperature not greater than 45”) to yield a white crys- talline solid. After drying over phosphorus pentoxide in oacuo, the aconic acid was washed out with petroleum ether (b.p. 30-60”). Yield, 245 mg.; m.p. 162-163”; specific radioactivity, 105.5 PC. per mole. In some degradations, the aconic acid was liberated with ethereal HCl, as described by Campbell and Hunt (15).

Degradation of Aconic Acid-Aconic acid (245 mg.) in water (12 ml.) was refluxed for 16 hours by using an oil bath at 110’. A stream of COz-free nitrogen was passed through the flask, and the effluent gas was bubbled through a saturated solution of barium hydroxide. The yield of barium carbonate was 264 mg. (71 per cent); the specific radioactivity of this barium carbonate sample (C, of itaconic acid) is shown in Table IT, “Degrada- tion 1.” 1 ml. of the remaining solution was t,reated wit,h p-nitrophenyl- hydrazine in 3 N HCl. The p-nitrophenylhydrazone of formylpropionin acid was obtained, m.p. 177-178”. To the remaining solution from the decarboxylation were added redistilled diethyleneglycol (12.5 ml.), hydra-


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zine hydrate (85 per cent, 2.5 ml.), and potassium hydroxide (0.5 gm.). The solution was refluxed for 1 hour, and sodium butyrate was then isolated as described by Moshach, Phares, and Carson (18). The yield of sodium butyrate was 73 mg. (39 per cent yield calculated from aconic acid, with allowance made for the aliquot removed in preparation of the p-nitrophenylhydrazone of formylpropionic acid). Schmidt degradations were carried out as described by Phares (19), and the radioactivity of the various barium carbonate samples is reported in Table II, “Degradation 1.”

TABLE II

Radioactivity of BaC03 Samples Obtained by Degradation of Itaconic Acid from 1-04-Glucose

BaCOa from carbon atom No.

Total radioactivity in BaC03 samples..............................

Radioactivity of itaconic acid used in degradation.

3pecific radioactivity, PC. per mole

legradatio 1

8.1 16.8 31.2 14.3 26.5

n

--

.-

1.5 8.4 3.2 17.3 4.9 32.2 1.9 14.7 5.2 27.4

96.9 16.7

102.0 18.2

Per cent total radioactivity

z%- 1

zx 2

8.9 19.2 29.4 11.4 31.1

verage of Degra- dations 1 and 2

8.6 18.3 30.8 13.0 29.3

Details of the culture conditions and the degradation are given in the text.

As a check, a dup1icat.e degradation was carried out in the same way by using a sample of the fermentation itaconic acid diluted with carrier to a specific radioactivity of 18.2 PC. per mole; the results of this degradation are shown in Table II, “Degradation 2.”

It was found in each case that about 80 per cent of the total radioactivity of itaconic acid obtained from I-Cl*-D-glucose was located in the 3 non-carboxyl carbon atoms. Co and C6 were almost equally labeled (average values, 30.8 and 29.3 per cent, respectively) and were significantly higher than CZ (average value, 18.3 per cent).

In the degradation of aconic acid obtained in the later experiments, the decarboxylation was carried out under the following conditions. Aconic acid (250 mg.) was dissolved in water (5 ml.) and diethylene glycol (12.5 ml.). The solution was heated for 2 hours at 160”, the CO2 being swept out in a stream of nitrogen. For the subsequent Wolff-Kishner reduction,


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the solution was treated with hydrazine hydrate (85 per cent, 2.5 ml.) and potassium hydroxide (0.5 gm.).

Incorporation qf I-P4-Acetate-A culture of A. terreus was grown for 7 days on growt,h medium (100 ml.) ; at this time it contained 6.0 mg. per ml. of itaconic acid. Sodium 1-Cr4-acetate (60 mg., 36.8 mc. per mole = 27 PC.) was then added, and the cotton plug replaced as previously described. Fermentation was continued for an additional 3 days, the con- centration of itaconic acid rising t’o 31 mg. per ml. On isolation and vacuum sublimation, 2.35 gm. of itaconic acid mere recovered; specific radioactivity, 650 PC. per mole (recovery of added Cl4 = 11.8 PC.; i.e., 43.7 per cent of that added). A sample of this material, diluted to a specific radioactivity of 132 PC. per mole, was degraded as previously described.

TABLE III

-

Radioactivity of BaC03 Samples Obtained by Degradation of Itaconic Acid from HWAcetic Acid

BaCOz from carbon atom No. Specific Per cent total radioactivity radioactivity

1

4

pc. per mole

58.8 71.6

Sum of radioactivity in BaC03 samples. 130.4 Radioactivity of itaconic acid used in degradation. 132.0

45.0 54.5

Details of the culture conditions and the degradation are given in the text.

The results of this degradation (Table III) show that the radioactivity was located almost exclusively in the two carboxyl groups.

Incorporation of Labeled Xuccinic Acid-The incorporation of labeled succinic acid during growth of A. terreus on non-radioactive glucose was also studied, since it seemed probable that the carbon atoms derived from a C4 dicarboxylic acid could then be traced without interference by label from Cs units. Extensive conversion of added succinic acid to acetic acid was not to be expected. Itaconic acid was therefore isolated after allowing 5 day cultures to stand overnight with either l-Cl4 or 2-C4-succinic acid. The itaconic acid isolated by crystallization (either from water or ethanol- petroleum ether) or by vacuum sublimation was contaminated by small amounts of a very radioactive material, probably unused succinic acid. This was also shown by the much higher specific activity of various samples of itaconic acid compared with that of recrystallized I-phenyl-2-pyrrolidinone-4-carboxylic acid, prepared from the samples by reaction with aniline. In these experiments, itaconic acid was therefore purified before degradation by partition chromatography. Celite columns moistened


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with 0.5 N sulfuric acid were used, with chloroform-10 per cent n-butanol as eluent (21).

Experiment I-2-C14-Succinic acid (22 PC., 13.24 mg.) in 3 ml. of sterile water was added to a 5 day-old culture grown on 100 ml. of growth medium. In this case aspiration was carried out by passing humidified air in a slow stream above a cotton plug as described by’ Corzo (22). After 18 hours, the culture contained 22 mg. of itaconic acid per ml. Recovery of COZ from the alkali absorber gave 2.13 gm. of barium carbonate, specific radioactivity, 57.2 PC. per mole; total recovery of Cl4 in COZ, 0.62 PC.

The crude itaconic acid obtained upon crystallization of the vacuum- concentrated culture medium had a specific radioactivity of 1000 PC. per mole. The total recovery of radioactivity in this material (contaminated with some highly radioactive succinic acid) was 16.9 ~c. The corrected value for the specific radioactivity of itaconic acid, purified by partition chromatography on a 75 gm. Celite column, was 230 PC. per mole. It was calculated that the recovery of radioactivity in the itaconic acid was 3.9 ye., corresponding to 18 per cent conversion of the added succinic acid. Degradation of 275.3 mg. of the pure itaconic acid, diluted to 1.012 gm. with carrier, gave the results shown in Table IV, Experiment 1.

Experiment 2-A 5 day-old culture of A. terreus was treated with 2-C14- succinic acid (22.4 PC., 14.53 mg.) for 22 hours. Itaconic acid, purified by partition chromatography, had a specific radioactivity of 248 PC. per mole, and was degraded in duplicate with use of the following dilutions: 346 mg. diluted to 1.000 gm. with carrier (Table IV, Experiment 2, Degra- dation 1); 226 mg. diluted to 830 mg. with carrier (Table IV, Experiment 2, Degradation 2).

Experiment S-l-C14-Succinic acid (29.9 PC., 12.60 mg.) in 3 ml. of sterile water was added to a 5 day-old culture. 20 hours later, analysis showed the presence of 16 mg. of itaconic acid per ml. of culture medium. CO2 samples were collected over various time intervals. For the first 1.5 hours, the specific radioactivity of the expired CO2 was 84.6 ~c. per mole. This value later approached a nearly constant level, being 221.0 PC. per mole for the period 1.5 to 4 hours, and 274.0 PC. per mole for the period 4 to 19 hours. The radioactivity recovered in CO2 (4.06 PC.) was 13.4 per cent of that added. The crude itaconic acid had a specific radioactivity of 1477 PC. per mole, corresponding to a recovery of 19.1 PC. The purified itaconic acid had a specific radioactivity of 290 PC. per mole; the total radioactivity in the itaconic acid was therefore 3.78 pc., corresponding to a 12.6 per cent conversion of the succinic acid to itaconic acid. The specific radioactivity of 1-phenyl-2-pyrrolidinone-4-carboxylic acid, prepared by reaction with aniline, was 286 PC. per mole, confirming the purity of the itaconic acid. For degradation (Table IV, Experiment 3), 339.5 mg. of pure itaconic acid were diluted to 997.6 mg. with carrier.


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Ml BIOSYNTHlGIS OF I’I’ACONIC ACID. I

All of these experiments with succinic acid as precursor show clearly that, by using 2-C14-succinic acid, CZ and Ca of itaconic acid became labeled equally (39 per cent of the total activity), with only small amounts of Cl4 in the other positions. By using 1-C14-succinic acid, the two car- boxy1 groups of itaconic acid (C, and C4) were labeled equally, with prac-

TABLE IV

Radioactivity of BaC03 Samples Obtained by Degradation of Itaconic Acid from Labeled Succinic Acids

BaCOa from carbon atom No.

Sum of radioactivity in BaCOa samples.

Calculated radioactivity in itaconic acid used in degradation..........................

Carbon atom No Per cent total radioactivity

--

C’4H.r COOH Experiment 1 Experiment 2 CWn.COOH A “Ha. COOH

L ‘“Hz. COOH Degr;dation Degra2dation

Ex eriment 3 Cl%. WOOH

L00013

Specific radioactivity, @cc. per mole

4.4 5.4 22.4 32.0 22.8 31.9

5.2 3.9 3.0 6.4

57.8 79.6

58.4 85.5

-

-.

-

7.6 6.8 16.6 50.6 38.8 40.2 40.8 39.4 40.0 39.2

9.0 4.9 5.3 49.4

5.2 8.1 8.1

4.1 25.6 24.6

3.3 5.1

52.2

50.6

62.7 102.8

67.5 98.6

Details of the culture conditions and the degradations are given in the text.

tically no Cl4 in other positions. In other words, it is apparent that the complete carbon skeleton of succinic acid is retained in itaconic acid.

DISCUSSION

Although earlier workers have concluded that cis-aconitic acid is not a precursor of itaconic acid (23, 24), we have prepared, from surface and shake cultures of A. terreus, soluble, cell-free extracts of the enzyme cis- aconitic decarboxylase, which rapidly decarboxylate cis-aconitic acid to equivalent amounts of itaconic acid and CO2 (25). For the purpose of


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this discussion, cis-aconitic acid will therefore be taken as the normal, immediate precursor of itaconic acid.

Evidence for C&J3 Split of Glucose-When the mold is grown on l-C14- glucose, the radioactivity of the expired CO2 is low initially, rising to an approximately constant value by the 3rd day of growth (see Table I). This is the opposite of the behavior observed with another Aspergillus mold, Aspergillus flaws, in which the Warburg-Lipmann-Dickens oxidative pathway of glucose dissimilation is predominant (10, 26). The progressive increase in radioactivity of the CO2 samples shows that the primary degradation of glucose does not involve C1. It is most likely, therefore, that the l-labeled glucose gives rise to methyl-labeled pyruvate by the Emb- den-Meyerhof reactions. The tentative proposal of Kinoshita (5, 6) that gluconic acid may be an intermediate in itaconic acid formation is unlikely in view of these observations.

Formation of Tricarboxylic Acid Cycle Intermediates-The expected isotope distribution in itaconic acid is shown in Figs. 2, A and B. For the moment, it is assumed that all of the citric acid formed is converted to itaconic acid without further oxidation. Oxalacetate formed solely through the Wood-Werkman reaction, and not in equilibrium with a symmetrical Cd dicarboxylic acid, would lead to itaconic acid labeled equally in C3 and

A 2 C*H2.COOH 1 I /OH

- 3c I'COOH 6

--z 2 C.COOH 1

1, C*H2. COOH 5 3 bH2 . COOH h

C%3.CO.COOH

i

2 C'H3.COOH

-

C"H2.COOH 2 I C"O.COOH 3 3

C'H3fCOOH b

dtH2.COOH 1 IJOH C" I,\COOH 6- C H;.COOH 5

c%2

b COOH 1 I - C*H2.COOH h

FIG. 2. Predicted labeling in itaconic acid derived from methyl-labeled pyruvic acid. A, oxalacetic acid formed by Wood-Werkman reaction and condensed directly with methyl-labeled acetate. B, oxalacetate in equilibrium with a C4 dicarboxylic acid, or formed by a CZ + CZ condensation.

Cg only. The observed results (Table II) show that Cz, Ca, and Cg contain most of the total radioactivity (78 per cent); Ca and Cs are equally labeled (30 per cent of the total radioactivity) and contain nearly twice as much activity as CZ (18 per cent). These observations suggest that most of the oxalacetic acid is formed by the Wood-Werkman reaction


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and is condensed directly with methyl-labeled acetate. Some oxalacetate, labeled in both methylene and ketone carbon atoms, must also be formed.

The mechanism suggested by Walker (8) would lead to equal labeling in C3 and Cs, and does not account for the appreciable label in CL. Fur- ther, it would not explain the observed rapid incorporation of acetate and succinate, and is therefore considered unlikely.

This preliminary conclusion is reinforced by the following argument, in which recycling of the tricarboxylic acids is no longer ignored. Shu et al. (27) degraded the labeled citric acid formed by A. niger from I-C14-

TABLE V

Comparison of Observed and CalclLlated Cl4 Distribution in Itaconic Acid Formed from I-P-Glucose

(1) (2) (3) -

6 1.37 1 3 3.99 2 4 6.71 3 5 * 1 4 2 6.71 5

Calculated per cent total radioactivity

Case A (4)

Case B (9

6.5 7.3 19.0 21.3 31.9 35.7 10.7 0.0 31.9 35.7

I-

-

Case c (6)

6.9 20.0 33.6 5.7

33.6

il

-

-

lo kserved per cent &xl radioactivity

(7)

8.6 18.3 30.8 13.0 29.3

* The radioactivity of Cg in citric acid is given the following values: Case A, 2.26; Case B, 0.0; Case C, 1.13. It is assumed that Cl of citric acid is lost as carbon dioxide during the action of cis-aconitic decarboxylase. If CF, of citric acid is lost, the distributions shown under Cases A, B, and C would be obtained with the following values for CE, in citric acid: Case A, 0.0; Case B, 2.26; Case C, 1.13. The radioactivity values in Column 2 are those given by Shu et al. (27) for citric acid obtained by growth of A. niger on I-C14-glucose; the calculated values in itaconic acid are derived from these values. The observed values (Column 7) are those obtained in the present work (see also Table II).

n-glucose, present at the time of spore inoculation, i.e. under conditions similar to those in our experiments. From this observed isotope distribution in citric acid, a theoretical isotope distribution in itaconic acid, formed by loss of a primary carboxyl group, may be calculated. The primary carboxyl groups of citric acid were not separated in the work of Shu et al., but initially it may be assumed that the radioactivity was equally divided in these carbon atoms. The calculated distribution is shown in Table V, Column 6, and is strikingly similar to that observed in our experiments (Table V, Column 7). The assumption that the primary carboxyl groups of citrate were equally labeled is not necessarily true, but since the activity in these groups is only 10.7 per cent of the total, any error is small. In an extreme case, Ch of citric acid would contain all of the activity. Ta- ble V, Column 4, shows the theoretical distribution in itaconic acid in this


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case, with loss of Cl of citrate; Table V, Column 5, shows the calculated distribution when Cg is not radioactive. In either case, the theoretical distribution is only slightly changed.

The excellent agreement between the observed and calculated isotope distributions in itaconic acid suggests that citric acid is formed in the same way by both A. terreus and A. niger. The conclusions of Shu et al., based on the mathematical analysis of the isotope distribution in citric acid, may be assumed to hold for the formation of the citric acid precursor of itaconic acid. These conclusions are that 78 per cent of the glucose is dissimilated by the Embden-Meyerhof pathway and that 60 per cent of the initially formed oxalacetic acid is derived by the Wood-We&man reaction and is labeled in the methylene group. In addition, about 40 per cent of oxalacetic acid, labeled in both methylene and ketone carbons, is formed. This was considered by Shu et al. to arise by a G + CZ condensation (Thunberg-Wieland) but could also be derived if oxalacetic acid were in equilibrium with a symmetrical Ch dicarboxylic acid by the reactions oxalacetate * malate ti fumarate, without cycling through citrate. How- ever, between 37 and 40 per cent of the Cg tricarboxylic acid is considered to be recycled to Cd dicarboxylic acid. The first two conclusions are in agreement with our earlier deductions.

Incorporation of Acetate-The results obtained with l-C14-acetate also agree with these general conclusions (see Table III). The labeled acetate was present over a 3 day period, and almost all of the Cl4 was located equally in the carboxyl groups. Using a different degradation from that described here, Corzo and Tatum (28) and Corzo (22) have studied the incorporation of 2-CY4-acetate into itaconic acid. The distribution pattern obtained in a long term experiment (13 days, shaken culture) was, as expected, similar to that reported here for 1-C14-D-glucose. In short term experiments (24 hours, shaken culture), these authors found 60 per cent of the radioactivity incorporated in the presence of 2-C14-acetate in Cs of itaconic acid. The major remaining activity (29.6 per cent) was in CZ and CB, which were not separately identified by their degradation. They suggested, therefore, that, in formation of itaconic acid, the carboxyl group of citric acid which was lost was that originally associated with acetic acid (C,), as shown in Fig. 3. The observation that most of the radioactivity in this experiment was located in one position (C, of itaconic acid) was presumably due to a decrease in the importance of recycling and other pathways for C4 dicarboxylic acid formation in the short term experiment.

Further evidence for the mechanism in Fig. 3 was obtained by Corzo and Tatum in an experiment (surface culture) in which asymmetrically labeled citric acid containing Cl4 in the two carboxyl groups derived from oxalacetat,e (C, and C,) was used. In this case, itaconic acid contained Cl4 largely in C1 and Cd.


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Our long term experiment with I-C14-acetate is still in agreement with this suggestion. Although citric acid formed from oxalacetate derived directly by the Wood-Werkman reaction will be labeled in only one car- boxy1 group (C,) and will consequently give rise to a non-radioactive itaconic acid, carboxyl-labeled C4 dicarboxylic acids can be formed by recycling of Ca tricarboxylic acids and possibly by the Thunberg-Wieland condensation. These reactions would lead to itaconic acid labeled equally in the two carboxyl groups (see Table III). Corzo and Tatum, in a 24 hour experiment with 1-C14-acetate, and using shaken culture, found 45 per cent of total radioactivity in C4 and 28.4 per cent in C1 of itaconic acid. The increased activity in Cq relative to C1 possibly resulted from COZ fixation by the Wood-Werkman reaction.

Incorporation of Xuccinate-It was apparent that the suggestion of Corzo and Tatum was unexpected if the double bond of cis-aconitic acid was formed between Cz and Ca of citric acid (as is the case with animal aconitase (29)) and if the subsequent decarboxylation removed the carboxyl

CH2. COOH 2 CH2. CCGH 1

I I

3 CH2. COOH 4

r-3 I

:o;13::,’ 1 b-H;:CO& 5’

2 C. COOH 1

5 [SH 2

FIG. 3. Formation of itaconic acid from 2-CWacetic acid

group attached to the secondary carbon of cis-aconitic acid. To gain further information, the experiments with succinic acid were carried out. It seemed unlikely that there would be extensive conversion of succinate to acetate, so that it would be possible to distinguish clearly the fate of an added C4 dicarboxylic acid. The anticipated, “normal” reactions (Fig. 4) would lead to the elimination of one of the carboxyl groups of succinic acid. If, however, the complete carbon skeleton of succinic acid was incorporated in itaconic acid, a different reaction sequence must have taken place. As shown in Table IV, both carboxyl groups of carboxyl- labeled succinic acid were retained in itaconic acid, and the Cl4 of methyl- labeled succinate was incorporated in Cz and CD of itaconic acid. The complete carbon skeleton of succinic acid is therefore retained during formation of itaconic acid, and the conclusion of Corzo and Tatum that the carboxyl group lost is that associated with acetate is fully confirmed. Further, the original supposition that there would not be much conversion of succinate to acetat-e is confirmed by the experimental observations. The radioactivity in Cg of itaconic acid was at most about, 8 per cent when methyl-labeled succinate was used.

Three possibilities may be considered to account for these results. (a) The action of mold aconitase may lead to the “abnormal” formation of


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the double bond of cis-aconitic acid between Ct and C., of citric acid. Such a reaction is apparently involved in the formation of glutamic acid in Clostridium Iclu~veri (30). Evidence for such a possibility might, be the known difference between mold and animal aconitase; although mold aconitase from A. niger has been resolved into two components (31, 32), it, has been concluded that animal aconitase is a single enzyme (33). (6) A ready explanation for the observed results with succinic acid would be the incorporation of added succinate and acetate into the tricarboxylic acid through the isocitritase reaction (34, 35) (Fig. 5). However, we have not, been able to detect isocitritase activity in extracts of A. terreus mycelium, and, in any event, this mechanism does not explain Corzo and Tatum’s result, with asymmetrically labeled citric acid. (c) The action of cis-aconitic

C*I$ CoOOH ,

C*H2. COOOH

/ ~.C"OO~l ~ $0'0" /2;. COOOH p

C*O. COOOH . COOOH -4~ Co. COOOH f I

LO C OOH CH3. COOH CH$SOOH %,. COOH !H,. CO@H

FIG. 4. Anticipated incorporation of succinic acid into itaconic acid. Carbon atoms of methyl-labeled succinate are distinguished as C*. Carbon atoms of carboxyl-labeled succinate are distinguished as C”.

CH3, COOH - CHO. COOH CH(OH). COOH CH. COOH *+o

C Hz. C OOH + h COOOH --+ 5. COOOH + I* C Hz. COOOH

I?:- * COOOH b

9 C Hp. 2. COOOH Hz. COOOH

FIG. 5. Incorporation of labeled succinic acids into itaconic acid via the isocitritase reaction. The symbols have the same significance as those in Fig. 4.

decarboxylase may remove the carboxyl group attached to the primary carbon atom of cis-aconitic acid, rather than the carboxyl group attached to the secondary carbon atom as has been generally assumed. Such a reaction mechanism would explain all of the known isotope distributions. Further, it is the mechanism that might, be expected in view of the known greater lability of unsaturated acids containing a P,y-ethylene bond with respect to compounds containing an cr,P-ethylene bond (36). From the present evidence, it, is not possible to make a final choice between these three possibilities; it will be shown in Paper III of this series (37) that the third possibility is the most, likely.

SUMMARY

The main mechanism for glucose dissimilation in itaconic acid formation by Aspergillus terreus is the Embden-Meyerhof pathway, as evidenced by the progressive increase in radioactivity of the carbon dioxide produced


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when the organism was grown on 1-CY4-n-glucose. Tracer experiments agree with Kinoshita’s hypothesis that itaconic acid is formed via citric and cis-aconitic acids. There is no evidence that the mechanism involves the condensation of 2 molecules of pyruvic acid, followed by dehydration and oxidative decarboxylation. About 60 per cent of the oxalacetic acid needed initially for citric acid formation is derived by the Wood-We&man reaction and is condensed directly with methyl-labeled acetate. The other 40 per cent of oxalacetic acid is either formed by the Wood-Werk- man reaction and is in equilibrium with a symmetrical Cq dicarboxylic acid or is formed by a Ct + Cz condensation. In addition, about 40 per cent of the citric acid is recycled to Cq dicarboxylic acid.

By using the two labeled succinic acids, it was found that the complete carbon skeleton of succinic acid was incorporated into itaconic acid. This would not be the case if cis-aconitic acid were formed by dehydration between the carbon atoms of the oxalacetate moiety of citric acid, and if the decarboxylation of cis-aconitic acid removed the carboxyl group attached to the secondary carbon atom. Possible explanations for the observed results are discussed. The one explaining all of the observations most readily is the assumption that the action of cis-aconitic decarboxylase is to remove the carboxyl group attached to the primary carbon atom of cis-aconitic acid.

BIBLIOGRAPHY

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Ronald Bentley and Clara P. ThiessenSUBSTRATES

STUDIES WITH C14-LABELEDASPERGILLUS TERREUS: I. TRACER

BIOSYNTHESIS OF ITACONIC ACID IN

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BIOSYNTHESIS OF ITACONIC ACID IN ASPERGILLUS TERREUS