BIOCHEMICAL STUDIES ON INOSITOL

III. BIOSYNTHESIS OF INOSITOL BY YEAST*

BY FRIXOS C. CHARALAMPOUS (From the Department of Biochemistry, School of Me&&e, University

of Pennsylvania, Philadelphia, Pennsylvania)

(Received for publication, August 14, 1956)

It is over a century since Scherer discovered in Liebig’s meat extract a hexahydroxycyclohexane which he named inositol (1). Since that time a number of similar compounds have been shown to occur in nature, and a few others have been synthesized in the laboratory. Of the nine possible stereoisomeric forms of hexahydroxycychlohexane, myo-inositol is by far the most widely distributed in nature and the one which has been impli- cated in the nutrition of animals and microorganisms (2, 3). It is because of its universal occurrence and its vitamin-like activity that inositol, among all other cyclitols, received the greatest attention by chemists and biochemists alike.

Among the many interesting problems concerned with the chemistry and metabolism of inositol, the mechanism of its biosynthesis still remains in the realm of speculation and uncertainty. That plants can synthesize inositol was quite evident from the beginning, since plants have been the richest source of inositol. Work by many investigators has established the fact that a number of microorganisms are able to synthesize inositol (4-7). With regard to the ability of animals to synthesize inositol, progress was slower since Vohl in 1858 reported that the urine of a man with diabetes insipidus contained inositol in amounts far in excess of the amount that could be accounted for from the food intake. Similar observations were later reported by Needham (8)) and others (9,lO) have shown that glucose- Cl* is incorporated into inositol in animals.

In a thorough presentation, Fischer discussed the chemical and biologi- cal relationships between hexoses and inositols (11). He proposed, as a working hypothesis, that inositol might be synthesized directly from glu- cose or glucose phosphates by a cyclization reaction. The observations of Portmann, as related by Fischer, that sharks have large amounts of inositol in the muscles of their fins, but no glycogen or other reserve carbohydrate in the liver, prompted the belief that inositol, formed from glucose directly, could serve as a source of blood glucose by the reopening of the inositol ring. The successful cyclization of glucose to inositol by chemical means by

*Supported by grant No. C-2228(C2) from the National Institutes of Health, Public Health Service, Bethesda, Maryland.

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596 BIOCHEMICAL STUDIES ON INOSITOL. III

Grosheintz and Fischer (11) offered a model experiment substantiating the cyclization hypothesis as the mechanism of its biosynthesis.

After the development of methods for the orderly chemical degradation of inositol as described in Paper II (12), it was felt that the problem of the biosynthetic origin of inositol could be studied with the aid of C4-labeled compounds. Furthermore, by using selectively labeled glucose-C*, the cyclization hypothesis could be tested in a more decisive manner.

The results presented in this paper indicate that in yeast glucose is not the immediate precursor of inositol and that smaller fragments, derived from glucose, condense to form inositol.

EXPERIMENTAL

Labeled Compounds-Glucose-l-C’*, glucose-2-C14, and glucose-6-C” were purchased from the National Bureau of Standards in Washington. 0*H&OONa and HC1400Na were obtained from Tracerlab, Inc.; HC4H0 from the California Foundation for Biochemical Research.

Culture Conditions-Torulopsis utilis Y-900 was grown with various radioactive compounds as described in Paper II (12). In the case of the cultures containing glucose as the sole source of carbon, each sample of radioactive glucose was diluted with unlabeled glucose to give a final con- centration of 2 per cent with the following specific activities (counts per minute per micromole of glucose) : glucose-l-Cl4 1806, glucose-2-Cl4 1908, glucose-6-C4 1748. In the case of the cultures containing C14H&OONa, HC400Na, or HC4H0, unlabeled glucose was added to give a final con- centration of 1 per cent. The acetate flask received 20 mg. of radioactive acetate containing 190 X lo6 c.p.m., the formate flask received 200 mg. of formate with 192 X lo6 c.p.m., and the formaldehyde flask received 6 mg. of formaldehyde containing 200 X lo6 c.p.m.

Isolation of Radioactive Inositol-At the end of 48 hours, the cells from each culture were harvested, and the inositol content was determined as described in Paper I (13). After the addition of carrier inositol the radio- active inositol from each culture was isolated (12) and chromatographed on Dowex 1, borate columns, followed by crystallization to constant specific activity. The purity of the isolated inositols was determined from (a) specific activity data before and after column chromatography, (b) paper chromatograms and specific activity determinations of the eluted inositol areas, and (c) specific activity data in which the amount of inositol was determined gravimetrically, as well as by the specific bioassay method (13).

Degradation ofInositol--Each sample of radioactive inositol isolated from the cultures grown with radioactive glucose was degraded by the procedure described in Paper II (12). The nn-idosaccharic acid was resolved into its optical isomers, and further oxidation with periodic acid produced the

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F. C. CHARALAMPOTJS 597

expected amounts of glyoxylic and formic acids. The glyoxylic acid was oxidized with HI04 to equimolar amounts of CO2 and HCOOH as described earlier, except that the reaction was carried out in test tubes instead of Warburg flasks. The COZ was swept with COz-free air into traps contain- ing NaOH solution and plated as BaC03. The formic acid produced from the oxidation of the idosaccharic and glyoxylic acids was isolated by distil- lation and plated as HCOONa or oxidized to CO2 and plated as BaC03. The quantitative aspects of the entire degradation have been described in detail in Paper II (12).

TABLE I Biosynthesis of Inositol-Cl4 by Yeast from Various Cl4-Labeled Substrates

1.05 0.60 1.39

Glucose-1-V. . 1,806 22.2 1900 Glucose-2-W. 1,908 23.8 1144 Glucose-6-C’*. 1,748 23.9 2426 CYH&OONa .._ :::: 780,000 23.0 1616 HPOONa 65,306 22.9 2616 HWHO 1,000,000 22.5 337

I I 100 mg. of carrier inositol added in each experiment. * Each culture flask contained 200 X lo6 c.p.m. For the amounts of the various

substrates added, see the text. t The specific activities have been corrected for the dilution by the carrier inosi-

to1. 1: This is the ratio of the specific activity of inositol to that of the corresponding

substrate.

Plating and Counting Methods-Organic compounds were plated on cop- per disks at infinite thinness, while samples of BaC03 were mounted on tared paper disks. All samples were counted in a windowless gas counter and corrected for self-absorption. The over-all counting error was 3 per cent.

Results

Table I summarizes the data with regard to the amount and specific activity of inositol synthesized from various radioactive substrates. The figures in the last column indicate the relationship between the specific activity of inositol and that of the corresponding substrate. It is clear that C-6 of glucose’ was incorporated into inositol much more extensively than

1 The abbreviations C-l, C-2, etc.. indicate carbon atoms 1. 2, etc., of glucose.

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598 BIOCHEMICAL STUDIES ON INOSITOL. III

either C-l or C-Z. The apparent incorporation of C-6 was 1.3 times that of C-l and 2.32 times that of C-2. The incorporation of HUJOONa and CY4H&OONa into inositol was very high indeed, giving rise to inositol with a specific activity comparable to that obtained with glucose-l-W and glucose-G-C4 in spite of the extensive dilution of the acetate and formate carbons by the unlabeled glucose present in the culture medium. The poor incorporation of formaldehyde into inositol may be partly due to the loss of formaldehyde during the aeration of the cultures. No attempt was made to calculate the “relative specific activity” of inositol when acetate, formate, or formaldehyde was the radioactive substrate, since these com- pounds were continuously “diluted” by similar compounds formed from the unlabeled glucose that was present. The radioactive inositols obtained from the cultures grown on glucose-Cl4 were degraded to nn-idosaccharic

TABLE II

Degradation to D- and L-Idosaccharic Acids of Labeled Inositols Synthesized from Glucose-P

Specific Inositol used in degradation specific Specific activity of

C’Gbeled substrate actwity of activity of idosaccharic acids isolated inosito1

degraded

C’Gbeled Unlabeled inositol D form I form

~~ C.).rn. per

pmole w. w. C.).??t. per c.p.m. per pm& p?Wk

G.).?n. per pmole

Glucose-1-P. . 345.6 90 180 115.2 112.8 114.7 Glucose-2-W. 224.7 40 180 40.9 42.0 40.9 Glucose-6-C14. . . . 466.6 90 180 155.5 156.0 153.2

acid after appropriate dilution with unlabeled inositol. The racemic acid was resolved into its optical isomers, and aliquots were plated and counted at infinite thinness. The results are summarized in Table II. The purity of each idosaccharic acid (racemic form) was determined by crystallizing its phenylhydrazide to constant specific activity, followed by regeneration of the free acid and crystallization of the acid potassium salt. The specific activity of the latter was indistinguishable from that of its corresponding phenylhydrazide. Approximately 25 mg. of each isomer of idosaccharic acid were obtained with the following specific rotations: for the three D

isomers [c# was +6.18”, +6.15’, +6.20” and for the three L isomers [a]~’ was -6.17”, -6.20”, and -6.18’.

50 pmoles of each isomer of idosaccharic acid were degraded to glyoxylic and formic acids, and aliquots of their sodium salts were plated and counted. In the case of formic acid another aliquot from each sample was oxidized to CO2 by HgC&, and the COZ was plated as BaC03 and counted. The counts obtained by direct plating of the formates were in good agreement

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F. C. CHARALAMPOUS 599

with the counts obtained from the corresponding BaC03 plates (variation less than 5 per cent). The results of this degradation are shown in Table III.

The distribution of radioactivity in the various samples of glyoxylic

TABLE III Degradation of Idosaccharic Acid to Glyoxylic and Formic Acids

C”-labeled substrate

Glucose-l-U4

Glucose-2-P

Glucose-6-P

Idosaa;dF T Acid formed Total radioactivity in acid

Glyoxylic Formic

pmozes /moles

96.95 98.3 99.21 97.8 98.9 100.0

100.2 97.7 98.75 97.9

101.30 98.4

Glyoxylic Formic

C.).rn. c.$b?L

3249 2280 1425 4104 999 1098

2097 0 5000 2801 3944 3840

* 50 Mmoles of each isomer of idosaccharic acid were used in the degradation, containing a total radioactivity of 5700 c.p.m. in the case of glucose-l-C**, 2100 c.p.m. in the case of glucose-2-W, and 7750 c.p.m. in the case of glucose-6-W.

TABLE IV Distribution of Radioactivity in Glyoxylic Acid

C’%beled substrate

Glucose-l-Cl4

Glucose-2-P

Glucose-6-U4

Type of glyoxylic acid-C”*

C.&m. C.).rn. c.p.m. D (3249) 0 3269 L (1425) 1405 0 D (999) 980 0 L (2097) 725 1361 D (%oo) 0 4936 L (3944) 4057 0

I Cl4 content of 2 carbon atoms of glyoxylic acid

co2 HCOOH

* The D and L letters refer to the D- and n-idosaccharic acids from which the re- spective glyoxylic acids were derived. The figures in parentheses indicate the total radioactivity of each sample of glyoxylic acid that was degraded.

acid was achieved by HIO, oxidation to HCOOH and COz. They were plated and counted as described above. The results are given in Table IV.

As a result of this degradation of inositol, its 6 carbon atoms have been separated into three distinct fractions; (a) the COZ fraction containing the two carboxyls of the two glyoxylic acids, (b) the formic acid fraction con- taining the two carbonyls of the two glyoxylic acids, and (c) the formic acid

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600 BIOCHEMICAL STUDIES ON INOSITOL. III

fraction containing the two formic acids derived from one idosaccharic acid. Since the sequence of the carbon atoms in the D- and n-idosaccharic acid is different, each of the three fractions mentioned above will contain

TABLE V Relationship of Carbon Atoms of Idosaccharic Acid to

Those of Glyosylic and Formic Acids I

Fragment derived from degraded idosaccharic acid

I-c-L-LCOOH lb5

(n-Idosaccharic kc?)

CO2 of glyoxylic acid.. . . . 1+2 2+3 HCOOH of glyoxylic acid.. . 3+6 1+4 Formic acid. . . . . . . . . . . . . . . . 4+5 5+6

IIOOC-L-c-L-c-COOH 2 id513

(L-Idosaccharic acid)

The numbers refer to the carbon atoms of inositol from which the idosaccharic acids were derived. See also Fig. 1 of Paper II (12).

TABLE VI Distribution of Radioactivity in Idosaccharic Acids and

Relationship to Carbon Atoms of Inositol

Carbon atoms of Cl’ content of various carbon atoms of idosaccharic acid fro& Idosaccharic acid inositol in idosaccharic

acid* Glucose-1-o Glucose-2-W Glucose-6-W

c.p.m. c.p.m. c.p.m.

D 1+2 0 669 0 3+6 1072 0 1555 4+5 752 629 874

L 2+3 470 415 1229 1+4 0 783 0 5+6 1354 0 1299

* See Table V. t The radioactivity of 1 pmole of idosaccharic acid obtained from inositol-Cl4 was

1898 c.p.m. in the case of glucose-l-C la,1173 c.p.m. in the case of glucose-2-W, and 2418 c.p.m. in the case of glucose-6-W.

different carbon atoms of inositol. This relationship is illustrated in Table V.

In those experiments in which the yeast was grown with glucose-U4 as the sole source of carbon, it is possible to calculate the contribution of glucose carbon atoms to the biosynthesis of inositol. To do this one needs to know the specific activities of the respective glucose and inositol, the dilution of the synthesized inositol by the unlabeled inositol added during its isolation and degradation, and finally the distribution of radioactivity

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F. C. CHARALAMPOUS 601

in the three fractions mentioned above. From the data presented in Tables I to V, one can calculate the data of Table VI. The dilution factor for the synthesized inositol-Cl4 was 16.5, 28.6, and 15.6 in the case of glucose-l-CY4, glucose-2-C14, and glucose-6-C14, respectively. -The data given in Table VI have been corrected for the above dilutions and were calculated for 1 pmole of inositol. From.these data it is possible to deter- mine the radioactivity of each carbon atom by considering each time a pair

TABLE VII Contribution of Glucose Carbon Atoms to Biosynthesis of Inositol

I !a-+rifir I C” content of carbon atoms of fnositol

inositol 1 2 ~--

c.p.m. per ).mo2e c.p.m. G.).#.

Glucose-l-U4 . . . . . . . . . . . . . . . . . . 1898 0 0 Glucose-2-W. . . . . . . . . . . . . . . . . . . 1173 154 415 Glucose-6-P. . . . . . . . . . . . . . . . . . . 2418 0 0

(0:08)2 2(0.21)

L-

3 4

I I

3 ---

C.p.f?S. c.p.n. c.p.m.

470 0 752 0 629 0

1229 0 j 874

-

.- ,

-

6

c.p.m.

662 0

326

(0.41) I z(O.32) (0.5016

FIG+. 1. Contribution of glucose carbon atoms to the biosynthesis of inositol. The numbers inside the ring indicate the carbon atoms of inositol, while those outside the ring indicate the glucose carbon atoms. The figures in parentheses indicate the activities of the various inositol atoms calculated as fractions of the corresponding glucose activities.

(D and L forms) of idosaccharic acids derived from the same inositol. For example, if one considers the D- and L-idosaccharic acids that were syn- thesized from glucose-l-04, one finds that the 470 c.p.m. obtained in carbon atoms 2 and 3 of the L isomer must reside in carbon atom 3, since carbon atoms 1 and 2 (obtained from the D isomer) were devoid of any activity. In a similar manner one can determine from the experimental values, the radioactivity of each carbon atom. The results are summarized in Table VII. The relationship of the glucose carbon atoms to those of inositol is better illustrated in Fig. 1 in which the activity of each inositol carbon is expressed as the fraction of the corresponding glucose activity.

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602 BIOCHEMICAL STUDIES ON INOSITOL. III

DISCUSSION

The results presented in this paper indicate that glucose is not the im- mediate precursor of inositol. This conclusion is based on the following considerations: (a) The specific activity of the synthesized inositol varied from 0.60 to 1.4 of that of the precursor glucose, depending on the type of glucose-U4 used. If randomization of Cl4 between C-l and C-6 of glucose occurred through the triose phosphates of the glycolytic pathway, one would expect equal specific activities of the inositols synthesized from glucose-l-P and glucose-6-CY4. The operation of the glucoseB-phosphate shunt coupled with the transaldolase and transketolase reactions would tend to dilute the “glucose pool” by the loss of Cl4 from glucose-l-U4 but not from glucose-6-C14. This would explain the finding that the specific activity of inositol from glucose-l-Cl4 was lower than that from glucose- 6-C14. However, this cannot explain the very low specific activity of ino- sitol obtained from glucose-2-C14. The operation of the above mentioned pathways would tend to preserve the C-2 of glucose to a greater extent than the C-l, with the result that the activity of the “glucose pool” would be higher in the case of glucose-2-Cl4 than in the case of glucose-1-CY4. (b) The fact that the specific activity of inositol obtained from glucose-B-U4 was significantly higher than that of glucose-6-C14 itself cannot be ex- plained from considerations of the pathways mentioned above if inositol was synthesized by cyclization of glucose. There is no simple explanation to account for the increase of the specific activity of glucose-6-C14 by con- sidering the metabolic pathways mentioned above. Any “metabolic intramolecular rearrangement” of the labeled carbon atom of glucose-6-Cl4 to produce glucose molecules with more radioactive carbon atoms should also produce equivalent molecules with less or no radioactivity; thus the specific activity of the glucose pool should remain fairly constant. It must be borne in mind that the yeast was grown on a medium with no other source of carbon than the added labeled glucose. (c) The distribution pattern of the glucose carbon atoms in the inositol molecule is even more convincing in implicating a biosynthetic mechanism other than cyclization of glucose. In a mechanism involving cyclization one expects to find the C-l and C-6 of glucose next to each other in the inositol molecule. This was not however the case. Both C-l and C-6 occupied the same positions in inositol. (d) The unequal contribution of C-l and C-2 of glucose to the biosynthesis of inositol is not anticipated from considerations of the met- abolic pathways mentioned earlier in the discussion if cyclization is the mechanism of inositol biosynthesis.

The present findings can be explained by a mechanism involving the condensation of a tetrose with a 2-carbon fragment. From the data of Fig. 1 it is evident that carbon atoms 6, 1, and 2 of inositol are derived

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F. C. CHARALAMPOUS 603

only to a small extent from C-l, C-2, and C-6 of glucose, and, therefore, they must have arisen from C-3, C-4, and C-5 of glucose. This, as well as the peculiar distribution of the C-2 of glucose, suggests that a tetrose, de- rived from glucose through the known transaldolase and transketolase

DIAGRAM 1 Suggested Pathway of Biosynthesis of Inositol from Glucose

Singly labeled glucose*

f&.-(y~c-c-~~-c* --

I (1) L-------,

I C4 C*6 C3 I I I

co5 c3 (2) c3 co5 c4 I I AI I I

C*6 C4 C” 5 co5 C” 5 a-Carbon pool I I I

!

C*6 C*6 C*6 Cl*-tetrose pool

(3) r--%4 --

Inositol c-c-c-c-c-c 4” 5* 6* 1" 2" 3*

Glucose carbon atoms

5” 6* 6* 5” 5” 6* 5” 6* 3 4 5’ 6* 5” 6* 4 3 5” 6*

_ * C-l and C-2 of glucose become C-6 and C-5, respectively, by randomization through the triose phosphates of the glycolytic pathway. Reaction 1 is catalyzed by transketolase and transaldolase. C” indicates glucose-2-W or glucose-5-W; C* indicates glucose-6-W.

reactions, contributes carbon atoms 3, 2, 1, and 6 of inositol, while a 2- carbon fragment contributes carbons 4 and 5. The 2-carbon fragment does not appear to be identical with that of the transketolase reactions, since C-l and C-2 of glucose contribute unequally to the inositol carbon atoms. A 2-carbon fragment derived from the tetrose mentioned above is suggested and could furnish the remaining 2 carbon atoms of inositol. The “tetrose pool” would be in equilibrium with the “Z-carbon pool,” and 1 mole of inositol would arise from equivalent amounts of the tetrose and

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604 BIOCHEMICAL STUDIES ON INOSITOL. III

the 2-carbon fragment. Such a concept, the qualitative aspects of which are illustrated in Diagram 1, can explain the observed distribution of glu- cose carbon atoms in inositol. It is indicated from Diagram 1 that the Cl4 content of the various carbons of inositol bears the following relation- ships: C-3 equal to C-5 and both 3 times larger than C-6; C-2 equal to C-4 and both 3 times larger than C-l. However, these relationships will be influenced by the relative rates of Reactions 2 and 3 (Diagram 1).

It was interesting to note that Posternak et al., following the metabolism of inositol labeled with deuterium in position 2, obtained glucose with the deuterium in C-6 (14). The authors concluded that, if synthesis of inositol proceeds through the same pathway as its degradation, cyclization of glucose could not be the mechanism since the deuterium was found in C-6 of glucose instead of C-5.

The suggested pathway for the biosynthesis of inositol bears some resemblance to that of shikimic acid (15). Unlike inositol, however, shi- kimic acid derives only 2 of its carbons from C-6 of glucose. Also the incor- poration of CY4-labeled acetate and formate in shikimic acid was poor com- pared to the high incorporation in inositol. However, both inositol and shikimic acid may share a common tetrose.

The detailed mechanism of the biosynthesis of inositol is now under investigation with cell-free systems.

SUMMARY

Inositol has been isolated from yeast cultures grown on glucose labeled with Cl4 in various positions and on C4-labeled acetate, formate, or form- aldehyde plus unlabeled glucose. The labeled inositols were degraded in a manner permitting the determination of the radioactivity of each carbon atom. The results obtained indicate that inositol is not synthesized directly from glucose by a cyclization reaction, but rather from smaller fragments derived from glucose. A pathway, involving the condensation of a tetrose with a 2-carbon fragment, was suggested as a possible biosyn- thetic mechanism.

It is a pleasure to acknowledge the expert technical assistance of Mr. Thomas Pattison throughout this work.

BIBLIOGRAPHY

1. Scherer, J., Ann. Clwm., 73,322 (1850). 2. Lardy, H. A., in Sebrell, W. H., Jr., and Harris, R. S., The vitamins, New York,

2, 342 (1954). 3. Woolley, D. W., J. Nutr., 28, 305 (1944). 4. Pope, H., and Smith, D. T., Am. Rev. Tuberc., 64, 569 (1946). 5. Jones, L. W., and Greaves, J. E., Soil SC., 66, 393 (1943).

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F. C. CHARALAMPOIX 605

6. Leonian, L. H., and Lilly, V. G., Science, 96, 653 (1942). 7. Lewis, J. C., Arch. Biochem., 4, 217 (1944). 8. Needham, J., Rio&em. J., 18,891 (1924). 9. Daughaday, W. H., Lamer, J., and Hartnett, C., J. Biol. Chem., 212,869 (1965).

10. Halliday, J. W., and Anderson, L., J.,BioZ. Chem., 217, 797 (1955). 11. Fischer, H. 0. L., Harvey Lectures, 40, 156 (194445). 12. Charalampous, F. C., J. Biol. Chem., 226, 585 (1967). 13. Charalampous, F. C., and Abrahams, P., J. BioE. Chem., 226,575 (1967). 14. Posternak, T., Schopfer, W. H., and Reymond, D., HeZv. chim. acta, 38, 1283

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Frixos C. CharalampousINOSITOL BY YEAST

INOSITOL: III. BIOSYNTHESIS OF BIOCHEMICAL STUDIES ON

1957, 225:595-606.J. Biol. Chem. 

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