on the mechanism of pyrimidine metabolism by yeasts knowledge of the mechanisms

ON THE MECHANISM OF PYRIMIDINE METABOLISM BY YEASTS

BY FREDERICK J. Dr CARLO, ALFRED S. SCHULTZ, AND ADRIENNE M. KENT

(From The Fleischmann Laboratories, Standard Brands, Inc., New York, New York)

(Received for publication, May 12, 1952)

Knowledge of the mechanisms involved in pyrimidine metabolism is fundamental to the biochemistry of the nucleic acids. Cerecedo, Emerson, and Stekol (l-7) pioneered this field in their work with dogs. Their experi- ments, conducted by feeding the in vitro oxidation products of uracil and the estimation of excreted urea, culminated in the proposal of a mechanism for pyrimidine metabolism (8). More recently, isotopically labeled cytosine was administered to rats and found to be catabolized partly to urea (9.

The present investigation extends the previous work on the metabolism of nucleic acid derivatives by yeasts (10) to provide more complete cover- age of pyrimidines and related compounds. The oxidative mechanism proposed by Cerecedo and his collaborators for the degradation of uracil into urea was found inoperative in yeasts. Unlike soil bacteria (ll-13), the yeasts were incapable of oxidizing uracil and thymine into barbituric acid and 5-methylbarbituric acid, respectively. On the basis of growth studies with yeasts the following metabolic pathway for the assimilat,ion of pyrimidines by yeasts is suggested: cytosine + uracil + hydrouracil + hydroorotic acid + urea.

EXPERIMENTAL*

The yeast cultures employed were Smcharomyces cerevisiae Hansen, re- quiring pantothenic acid and biotin as growth factors (14), and To&a utilis (Northern Regional Research Laboratory). The techniques for t.he preparation of the yeast inocula and the measurement of yeast growth were described previously (14). The composition of the basal medium was also reported earlier (15).

The yeasts were incubated at 30” with shaking in 18 mm. (outside diameter) test-tubes and the extent of growth was determined after 16, 24, and 40 hours by reading the percentage light absorption on a Lumetron 400 calorimeter equipped with a gray glass and wire screen filter. Growth data acquired for S. cerevisiae Hansen and T. utilis propagated on ammon-

1 The microanalytical work was performed by Mr. Joseph F. Alicino, Metuchen, New Jersey.

333

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334 PYRIMIDINE METABOLISM BY YEASTS

ium sulfate as the sole source of nitrogen were published earlier (10). Each compound tested was furnished to the yeasts in a quantity corresponding to 1.0 mg. of nitrogen per tube. The compounds were used as sole nitrogen sources and also with supplementation by 0.1 mg. of nitrogen in the form of ammonium sulfate. The results presented are based upon the data obtained from at least six tubes.

TABLE I

Compounds Not Assimilated by Either Yeast Culture

Acetylurea

Acrylamide Allylurea 2-Amino-4-methyl-6-chloropyrimidine 2-Amino-4-methyl-6-hydroxypyrimidine 2-Aminopyrimidine 5-Aminouracil Barbituric acid Biuret Cyanoacetamide 2,5-Dimethyl-6-hydroxypyrimidine Folic acid Hydantoic acid Hydantoin Hydrouracil-5-carboxylic acid 2-Hydroxypyrimidine Isobarbituric acid Isocytosine Isodialuric acid Malonamide 2-Methyl-5-aminomethyl-6-aminopyrim-

idine 4-Methylcytosine 2-Methyl-5-cyano-6-aminopyrimidine 2-Methyl-5-ethoxymethyl-6-aminopyrim-

idine

-

-

2Methyl-5-ethoxymethyl-6-hydroxypyrimidine

6-Methylthiouracil 6-Methyluracil Methylurea 5-Nitrobarbituric acid Orotic acid Oxamide Oxamic acid Oxythiamine Pteroic acid Semicarbazide Succinamic acid Succinimide Succinylurea Thiamine Thiouracil Thiourea Thymine

‘I glycol Uracil-5-carboxylic acid Uramil

Urethane Ureidosuccinic acid Xanthopterin

-

Compounds which were not utilized as nitrogen sources for growth by either S. cerevisiae or T. utilis are listed in Table I. The growth data obtained with compounds serving as nitrogen sources for one or both yeasts are shown in Table II.

Hydrouracik-2.24 gm. (0.02 mole) of uracil were dissolved in 200 ml. of hot glacial acetic acid. After the solution had cooled to room temperature, 300 mg. of Adams’ catalyst were added. The mixture was hydrogenated at about 25” under an initial pressure of 40 pounds per sq. in. When the


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F. J. DI CARLO, A. S. SCHULTZ, AND A. M. KENT 335

reduction was complete (5.5 hours), the catalyst was removed by filtration and the fikrate was poured illto 500 ml. of hesanc. The white precipitate collected by filtration was washed thoroughly with hexane, dried, and recrystallized from water. The product weighed 1.07 gm. and melted at, 275-276”. Brokn and Johnson (1G) reported a melting point of 272- 274”; Lengfeld and Stieglitz (17) reported 272”. A mixed melting point with an authentic sample of hydrouracil obtained from Dougherty Chem- kals showed no depression.

Pofnssiwn /3-Ureidopropion&e-This compound was prepared from /3-

TABLE II

Compounds Assimilated by T. utilis and S. cerevisiae

Compound

____--__- -~ ~__. - ___-

.4cetamide ................................. Acetamidinc .............................. @-Alanine .................................. (3-.4minopropionamidc ...................... Asparagine ................................ Aspart,ic acid. ............................. Cytosine .................................. Ethyl @-aminopropionate ................... Formamide ................................ Hydroorotic acid. ......................... Hydrouracil............................... 5-Methylcytosinc . ......................... Oxaluric acid .............................. Propionamide .............................. Uracil..................................... p-Ureidopropionic acid .....................

Per cent nitrogen utilized

S. cerevisiae T. utilis

100 loo 95 95 90 95 95 95 90 95

100 35 90 90 95

100

alanine and potassium cyanate by the method of Lengfeld and Stieglitz (17).

l

Ureidosuccinic Acid-This compound was synthesized from aspart,ic acid and potassium cyanate as described by Nyc and Mitchell (18).

Hydroorotic Acid--This compound was prepared from maleic acid and urea according to the procedure of Rachstez and Cavallini (19).

CsHsOaNt. Calculated. C 37.97, H 3.79, N 17.72 Found. “ 37.93, ‘I 4.01, I‘ 17.92

&Aminopropionamide Hydrochloride-The method of Carlson (20) was modified. 840 mg. (0.01 mole) of cyanoacetamide were dissolved in 100 ml. of glacial acetic acid and hydrogenated for 3 hours in the presence of


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200 mg. of platinum oxide at an initial pressure of 38 pounds per sq. in. The catalyst was filtered off and washed with about 10 ml. of glacial acet.ic acid. The combined filtrate and washings were concentrated to about 25 ml. by vacuum distillat?on. To the concentrated solution were added 1.0 ml. of concentrated HCl and 300 ml. of hexane. The mixture was refrig- erated for several hours before collecting the precipitat,ed hydrochloride. The material was dissolved in slightly more than the minimum volume of water. Then concentrated HCl was added to pH 1. Alcohol (about 100 ml.) was added, followed by ether to produce turbidity. The white prc- cipitate collected after refrigeration weighed 815 mg. and melted at 146” (Franchimont and Friedmann (21) reported a mehing point of 149”).

C,HpON&l. Calculated, N 22.49; found, N 22.64

Succinylurea-Urea and succinic anhydride were fused according to the method of Pike (22). The product melted at 230” after softening at about 200”. Pike reported 203-205” as the melting point.

CHON 1 8 1 I. Calculated, N 17.59; found, N 17.52

Hydrouracil-&carboxylic Acid---Uracil-5-carboxylic acid (130 mg.) was dissolved in 80 ml. of warm glacial acetic acid. After the solution cooled to room temperature, 200 mg. of platinum oxide were added and the mixture was hydrogenated for 6 hours at a pressure of 50 pounds per sq. in. The catalyst was removed by filtration and the filtrate, suitably diluted, was found to contain no starting material by ultraviolet spectro- photometry. The addition of 600 ml. of hexane to the filtrate produced a precipitate which was collected after refrigeration for several hours. The dried product weighed 104 mg. It was found to darken above 235” and to melt at 255-256”.

CsH601N2. Calculated, N 17.72; found, N 17.95

RESULTS AND DISCUSSION

Hahn and Haarmann (23) discovered the abilitd of yeast autolysates to convert cytosine into uracil and 5-methylcytosine into thymine. Chargaff and Kream (24) prepared cell-free extracts of yeast which hydrolyzed cytosine into uracil. Their findings were substantiated by our previous work (10, 25), in which it was demonstrated that X. cerevisiae utilized only 1 nitrogen atom of cytosine for growth and was incapable of any growth on uracil, the corresponding deaminated compound, and that both S. cerevisiae and I’. utilis were able to assimilate 1 nitrogen atom of 5-methylcytosine, but no nitrogen from thymine. The specificity of cytosine deam- inase was evident from the failure of either yeast to grow on amino nitrogen


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from 4-methylcytosine, 2-aminopyrimidine, isocytosine, and other amino- pyrimidines listed in Table I.

The mechanism proposed by Cerecedo et al. for the metabolism of uracil in dogs is illustrated in Diagram 1. The initial attack upon uracil was represented as oxidation at the 5 position to yield isobarbituric acid. This was not the manner of uracil metabolism by yeasts. T. utilis, a yeast culture which assimilated all uracil nitrogen, was unable to utilize any nitrogen from isobarbituric acid. The further finding that T. utilis did not assimilate barbituric acid showed that the 4 position of uracil was not oxidized by this yeast as it was in soil bacteria (11-13). The failure of tither isodialuric acid or alloxan to promote growth of T. utilis eliminated t,he possibility of simultaneous oxidation at the 4 and 5 positions of uracil.

NH-CO NH-CO NH-CO NH-CHO IllI II I

CO CH+ CO COH+ CO CO + co -+ I II I II I I I

NH-CH NII-CR NH-CHOW NH-CO-COOH Uracil Iwhrbituric Isodialuric Formsloxaluric acid

acid acid

NH2 NH? I I COOH

CO I

-+ co + 1 I cooIr

NH-CO-COOB NH* Oxaluric acid Urea Oxalic

acid

DIAGRAM 1. Mechanism of uracil metabolism iu dogs proposed by Cerecedo et al.

These observations were regarded as precluding oxidation as the initial rcact,ion of uracil catabolism by yeast.

(tt)oxylation was eliminated as the first step in the assimilation of uracil because of the inability of both yeasts to grow on uracil-5-carboxylic acid or on erotic acid (uracil-4-carboxylic acid), a naturally occurring compound (26) utilized for pyrimidine biosynthesis in rats (27, 28), in slices of rat liver (29), and in Lamkh.cillus bzdgaricus (30).

The cnzymat ic hydrolysis of uruc*il was consid(~red possible at, threcl points, namely, at the 1,2, the 2,3, and the 1,6 amide linkages. Hydrolysis at either the 1,2 or the 2,3 position of uracil would yield unstable carbamic acid derivatives, both of which would be converted into /3-aminoacrylamide by decarboxylation. No synthesis of this compound has been devised. Malonamide, which differs from /3-aminoacrylamide only by it,s possession of an additional atom of oxygen, was tested. It supported no growth of cithcr yettat. Acrylamide was also useless as a nitrogen source. Hydroly-


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tic cleavage of uracil at the 1,6 linkage would yield 8-ureidoacrylic acid, another unknown compound. Hence we were unable to eliminate the possibility of uracil utilization by a mechanism involving hydrolytic cleavage.

Attention was turned to consideration of reduction as the step initiating uracil catabolism. Hydrouracil was tested and found, as uracil, to be assimilated completely by T. utibis, but to support no growth of S. cerevisiae. This finding led to scrutiny of possible metabolic degradation products of hydrouracil. Hydrolytic cleavage at the 1,2 or the 2,3 position was re-

HZN COO11 I I

OC CHZ I I

HN-CHn &Ureidopropionic acid

t HN-CO HN-CO H,N-CO

I I I I I OC CHZ +OC CH*--, CH,

I I I I IIN-CHCOOH HN-CH, HZN-CH2 Hydroorotic acid Hydrouracil @-Aminopropion-

1 amide

HN-CO I I

OC CHCOOH I I

HN-CH2 IIydrouracil-5-carboxylic acid

DIAQRAM 2. Investigated pathways of hydrouracil catabolism

gartled as leading to the formation of p-aminopropionamide via the uustablc carbamic acid products. Hydrolysis of hydrouracil at the 1,6 position would yield fl-ureidopropionic acid. Enzymatic carboxylation of hydrouracil could produce either hydrouracil-4.carboxylic acid (hydroorotic acid) or hydrouracild-carboxylic acid. These possible conversions arc shown in Diagram 2.

The four possible degradation products of hydrouracil were synthesized and tested. Hydrouracil-5-carboxylic acid did not support growth of either yeast and thus was eliminated as a catabolic product of hydrouracil which supported full growth of T. utilis. fl-Ureidopropionic acid and p-aminopropionamide served as excellent nitrogen sources for T. u[ilis, but per- mitted no growth of S. cerevisiae. It is suggested that both compounds


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P. J. DT PARTAl, .\. S. SSC:lIUl,TZ, AND A. M. KENT 330

were converted into @-alanine which has the same pattern of assimilation, namely, complet’e utilization by T. ulilis and no utilization by S. cerevisiae. Hydroorotic acid was found to support full growth of both yeast cultures. This result was of particular significance, since it represented the only instance in which the nitrogen bound in a pyrimidine ring was assimilatrtl by S. cerevisiac. <Jytosine and 5-methylcytosinc supported growth of S. cerevisiue, but only to the estent afforded by their free amino groups (10, 25).

The pathways investigated in the catabolism of hydroorotic acid arc shown in Diagram 3. They consisted in hydrolysis at the 1,G position to ureidosuccinic acid, reduction at the 3,4 position to succinylurca, :LII~ hydrolysis at either the 1,2 or 2,3 position to form asparagine after de-

&N-CO HN-CO 11,N COO11 I I I I I

CIIZ t oc CIIZ 4 oc GIL I I I I I

IIzN-CHCOOII HN-CHCOOH HN-CHCOOH Asparagine Hydroorotic acid Ureidosuccinic acid

HN-CO I I

OC CHz I I

HIN CH,COOH Succinylurea

DIAQRAM 3. Investigated pathways of hydroorotic acid catabolism

carboxylation of the unstable carbamic acids. Ureidosuccinic acid, an acyclic pyrimidine precursor in L. bulgaricus (30), was not utilized for growth by either S. cerevisiae or T. utilis, and was ruled out as the catabolic product of hydroorotic acid. Succinylurea also failed to support the growth of either yeast culture. Asparagine, however, was an excellent nitrogen source for both yeasts.

Asparagine was known to inhibit the growth of S. cerevisiae when pantothenic acid was replaced by /3-alanine (31, 32). The data presented in Table III showed that hydroorotic acid was not inhibitory under identical conditions. This evidence served to eliminate asparagine as the catabolic product of hydroorotic acid.

It was known that S. cerewisiae Hansen required considerably more biotin for growth on urea (33) or on compounds converted into urea than was necessary for comparable growth on other nitrogen sources.2 The relative dependence of the assimilation of nitrogen from ammonium sulfate,

* Di Carlo, F. J., Schultz, A. S., and Kent, A. M., unpublished work.


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urea, hydroorotic acid, and asparaginc upon the biotin concentration of the growth medium is illustrated in Fig. 1. The high biotin level needed for good growth on hydroorotic acid indicated that this compound was catabolized to urea. Asparagine was an effective nitrogen source at low biotin concentrations; obviously its assimilation was not routed through urea. The mechanism of asparagine assimilation was regarded M involving conversion into aspartic acid by t,he action of asparaginasc, an enzyme found

TABLE III Growth of S. cerevieiae on Asparagine, Hydroorotic Acid, and Urea in Presence of

Pantothenic Acid and &Alanine

N as (N&)rSOI Test compound’

w. 2 4

2

2

2 2 4

2

2

2

Asparegine “

Hydroorotic acid I‘ I‘

Urea I‘

Asparagine I‘

Hydroorotic acid I‘ “

Urea I‘

_-

-

B-Alanine, y per tube

1.7 1.7 1.7 1.7 1.7 1.7 1.7 1.7

l-

-

Growtht after

16 hn. 4C hn.

8.7 12.8 9.4 13.5 6.5 10.5

11.0 13.5 4.1 10.7 8.0 13.0 4.9 10.8 9.0 13.5 8.6 12.6 9.0 13.5 6.6 10.5 0.7 0.8 3.9 10.4 7.0 12.6 4.7 10.8 8.5 13.0

* Furnished to the extent of 1.0 mg. of nitrogen per tube. t The growth data are expressed in terme of optical density X 10.

in yeast by several groups of investigators (34), and the release of ammonia from aspartic acid to form fumaric acid. Aspartase, the enzyme which effects the latter conversion, was reported present in yeast autolysates (35). This interpretation was supported by the present findings that, whereas aspartic acid was an excellent nitrogen source for both yeast cultures, succinamic acid, obtainable from asparagine by deamination, supported no growth of either culture, and j3-aminopropionamide, obtainable by decarboxylation of asparagine, supported growth of only T. utilis.

The preceding considerations led to the proposal of a metabolic pathway for the assimilation of pyrimidines by yeasts. The scheme is presented in Diagram 4.


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x8

I I I I

5 IO I5 20 BIOTIN, M 7

FIG. 1. Effect of biotin concentration upon the extent of growth of S. cerevisiae on asparagine (Curve l), ammonium sulfate (Curve 2), urea (Curve 3), and hydro- erotic acid (Curve 4) after 24 hours.

N=CNHz HN-CO HN-CO

I I + II*0 I I I I OC CH

--cytosine deaminasc + oc CH +2HOC CHz

+ co::

I II I II I I -+ HN--CH HN-Cl1 HN-CHz

Cytosine Uracil Hydrouracil

HN-CO NIT*

I I I OC CHz + co

I I I HN-CHCOOH NH*

Hydroorotic acid Urea

DIAQRASI 4. Mechanism proposed for the catabolism of pyrimidines by yeasts


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The authors are grateful to Professor 1,. R. Cerecedo of Fordham Uni- versity for samples of isobarbituric acid, isodialuric acid, thymine glycol, and erotic acid, to Dr. Morris Soodak of the Massachusetts General Hos- pital for a sample of oxythiamine, and to Dr. George H. Hitchings of the Wellcome Research Laboratories for a sample of uracil-Bcarboxylic acid.

SUMMARY

1. The assimilation of nitrogen from 64 compounds by l’orula utilis and Saccharomyces cerevisiae Hansen was investigated. T. utilis utilized prac- tically all of the nitrogen from fifteen of these compounds, whereas only six supported the growth of S. cerevisicze.

2. T. utilis assimilated all of the nitrogen of uracil for growth, but failed to grow on isobarbituric or isodialuric acid. This showed that the yeast did not catabolize uracil by the pathway followed in dogs.

3. A mechanism for the catabolism of pyrimidines by yeasts was suggested from structural considerations and growth data.

4. The conventional scheme of asparagine catabolism received support ill this work.

BIBLIOGRAPIIY

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11. Hayaishi, O., and Kornberg, A., J. Am. Chem. Sot., 73, 2975 (1951). 12. Wang, T. P., and Lampen, J. O., J. Biol. Chem., 194, 775, 785 (1952). 13. Hayaishi, O., Federation Proc., 11, 227 (1952). 14. Schultz, A. S., and Atkin, L., Arch. Biochem., 14, 369 (1947). 15. Schultz, A. S., and Pomper, S., Arch. Biochem., 19, 184 (1948). 16. Brown, E. B., and Johnson, T. B., J. Am. Chem. Sot., 46,2702 (1923). 17. Lengfcld, II’., and Stieglitz, J., Am. Chem. .I., 16, 516 (1893). IS. Nyc, J. F., and Mitchell, II. K., J. Am. Chem. Sot., 69, 1382 (1947). 19. Bachstez, IQ., and Cavallini, G., Ber. them. Ges., 66 B, 681 (1933). 20. Carlson, G. H., U. S. patent 2,354,909 (1944); Chem. Abstr., 38, 6302 (1944). 21. Franchimont, A. P. N., and Friedmann, H., Rec. trau. chim. Pays-Bas, 26, SO (1906). 22. Pike, W. H., Ber. them. Ges., 6, 1104 (1873). 23. Hahn, A., and Haarmann, W., 2. Biol., 86, 275 (1926). 24. Chargaff, E., and Kream, J., J. BioZ. Chem. 176,993 (1948). 25. Di Carlo, F. J., Schultz, A. S., and Kent, A. M., J. BioZ. Chem., 194, 769 (1952).


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F. J. DI CARLO, A. S. SCHULTZ, AND A. MM. KENT 343

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Adrienne M. KentFrederick J. Di Carlo, Alfred S. Schultz and

METABOLISM BY YEASTSON THE MECHANISM OF PYRIMIDINE

1952, 199:333-343.J. Biol. Chem.

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