Biosynthesis of cyclopentenyl fatty acids

Biosynthesis of cyclopentenyl fatty acids

261 Biochimica et Biophysics 0 Elsevier/North-Holland Acta, 450 (1976) Biomedical Press 261-265 BBA 56667 BIOSYNTHESIS OF CYCLOPENTENYL FATTY A...

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Biochimica et Biophysics 0 Elsevier/North-Holland

Acta, 450 (1976) Biomedical Press


BBA 56667





UDO CRAMER Institut (Received


fiir Biochemie, May 26th,


SPENER Miinster,


23a, 4400 Miinster

(G. F. R.)


Summary The biosynthesis of cyclopentenyl fatty acids from (2cyclopentenyl)car boxylic acid (aleprolic acid) via chain-lengthening by Cz-units was tested in seeds and leaves of Caloncoba echinata and Hydnocarpus anthelminthica of Flacourtiaceae and in various preparations of higher plants other than Flacourtiaceae. Only tissues of Flacourtiaceae, where cyclopentenyl fatty acids are found naturally, were able to accept aleprolic acid as a starter molecule for the synthesis of cyclic fatty acids. Labelling patterns of straight chain and cyclic fatty acids, synthesized after incubation of Flacourtiaceae seeds with [1-‘4C] acetate, indicated de novo synthesis of Cl6 fatty acids in either case, followed by elongation to higher homologs. Introduction From the biogenetic point of view, chaulmoogric acid and other members of the cyclopentenyl fatty acid family pose an interesting problem. These acids, occurring exclusively in Flacourtiaceae of higher plants [l] , are not synthesized via cyclization of appropriately structured polyunsaturated fatty acids [2-41, as are the prostaglandins [5-71. Recently, a hypothesis was advanced that a small cyclic precursor may serve as a primer for the synthesis of cyclopentenyl fatty acids via chain-lengthening. Indeed, (2-cyclopentenyl)carboxylic acid (aleprolic acid) was lengthened to cyclopentenyl fatty acids by Chlorella vulgaris, a model system, which normally does not synthesize these acids [8].

* The results presented

are parts of U. Cramers’



In the course of the work reported here, aleprolic acid was tested as a precursor for cyclopentenyl fatty acids in the systems proper, i.e. seeds and leaves of Caloncoba echinata and Hydnocarpus anthelminthica of Flacourtiaceae. Included are studies with various organs and subcellular preparations of higher plants other than Flacourtiaceae, in order to test the potential of aleprolic acid as a general primer in fatty acid biosynthesis. Materials and Methods Radiochemicals

and cofactors.

[l i4C]Acetate (spec. act. 1 Ci/M) was obtained from New England Nuclear (Dreieichenhain, Germany) and [l-i4C] aleprolic acid (spec. act. 10 mCi/mM) was prepared as reported elsewhere [S]. COA, ATP, NADPH and NADH were purchased from Boehringer (Mannheim), and dithiothreitol from Sigma (Munich). Plants. Seeds and leaves of C. echiizata were received from the Belmonte Arboretum of the Landbouwhogeschool, Wageningen, The Netherlands and those of H. anthelminthica from Honolulu Botanic Gardens, Honolulu, Hawaii, U.S.A. Seeds of Corylus auellunu (Betulaceae), as well as leaves of Pyrus communis (Rosaceae) and Spinacia oleruceue (Chenopodiaceae) were obtained locally. Incubations of substrates and isolation of products. Leaves were chopped and incubated in 1 g batches as described by Yano et al. [9]. After 6 h, the medium was removed and leaf discs were homogenized in 80 ml isopropanol and 50 ml CHC13/CH30H (2 : 1, v/v) per sample. Lipids were extracted following established procedures [lo] and transesterified in 5 ml of CH,OH/benzene/ cont. HzS04 (90 : 10 : 5, by vol). Methyl esters were first purified by thin layer chromatography (Silicagel H, hexane/diethyl ether, 90 : 10 v/v) and than separated according to structural features by argentation thin layer chromatography (Silicagel G + 5% AgN03, hexane/diethyl ether, 80 : 20 v/v, 4°C). Upon elution from the adsorbent, the radioactivity incorporated into each fraction was determined in aliquots, followed by characterization of individual methyl esters via radio-gas-liquid chromatography, as described earlier [ 81. Intact chloroplasts were isolated and incubated according to the procedures of Kannangara and Stumpf [11,12]. Chlorophyll was determined by the method of Bruinsma [13]. Incubations were stopped after 1 h by addition of 10% KOH in 90% CH30H. Methyl esters were prepared and determined as reported [81.

Seeds were shelled, except those of C. echinata, halved and incubated for 6 h by the method’of Appleby et al. [14]. After removal of the medium, each tissue was homogenized in a total of 70 ml CHCl,/CH,OH (2 : 1, v/v). Preparation of methyl esters and determination of radioactivity was carried out as outlined for leaf lipids. Schmidt-degradation of labelled fatty acids. Cldecarboxylation of fatty acids was carried out according to Aronsson and Giirtler [15]. Yields were constantly 10% too low, and results presented in Table II are corrected for that value. The figures in Tables I and 11 represent the mean of 2-4 determinations each.


Results All tissues exhibited a good capacity for fatty acid synthesis, as shown by the incorporation of [l-14C]acetate into fatty acids. However, only the seeds and leaves of Flacourtiaceae were capable of synthesizing cyclopentenyl fatty acids from [1-14C] aleprolic acid (Table I), whereas C. aue2Zarza and P. communis did not accept this substrate. The same was true with chloroplasts of Sp. oleracea. Addition of inactive aleprolic acid did not interfer with the incorporation of [l-14C] acetate into chloroplasts. In seeds of C. echinata and H. anthelminthicu, [l-14C] aleprolic acid was incorporated almost exclusively (96.5 and 99.6%, respectively) into cyclopentenyl fatty acids. In contrast, [1-14C] acetate labelled mainly straight-chain fatty acids, although cyclopentenyl fatty acids comprise between 80 and 90% of the total seed fatty acids [Z]. Only 16.4 and 17.9% of the activity respectively, was found in cyclic acids. Data on the incorporation of substrates into leaf fatty acids of Flacourtia: ceae are less clear cut. A detailed analysis of labelled fatty acids revealed that only 2.2% of the radioactivity was found in cyclopentenyl fatty acids of C. echinutu leaves after incubation with [ l-14C] aleprolic acid and 0.5% after incubation with [l-14C]acetate. Similar results were obtained with leaves of H. anthelminthicu. Obviously, degradation of [ l-14C] aleprolic acid to acetate must have occurred; the acetate was then reused for de novo synthesis of straightchain fatty acids mainly. Degradation of the substrate could not be avoided by





COllCIl. WI)


Incorporation into total fatty acids (nM/g tissue)


Radioactivity in cyclopentenyl fatty acids w

C. echinata, mature seeds

aleprolic acid acetate

0.2 2.0

0.9 15.8

96.5 16.4

H. anthelminthica, immature seeds

aleprolic acid acetate

0.2 2.0

1.4 27.3

99.6 17.9

C. echinata, chopped leaves

aleprolic acid acetate

0.6 7.9

5.7 * 573.0

2.2 0.5

H. an thelmin thica, chopped leaves

aleprolic acid acetate

1.0 5.0

0.9 * 49.0

2.6 0.4

c. awllana, immature seeds

aleprolic acid acetate

0.2 2.0

0 126.0

0 0

P. communis, chopped leaves

aleprolic acid acetate

0.7 5.0

0 93.0

0 0

sp. oleracra. chloroplasts

aleprolic acid acetate

0.3 1.0

0 34.7 **

0 0

* Values include incorporation of substrate and of its degradation products (see text). ** nM/mg chlorophyll.














(% Total








Hydnocarpic (C16) Chaulmoogric (ClS) Hormelic








Il. an thetminthica (%



Theory (%

















43.4 acid

(C16) (C18) Arachidic



(C20) Palmitic acid Stearic



acid acid


shortening the incubation time to 0.5 h, or by preincubation with glucose, bicarbonate and acetate. However, the role of aleprolic acid as a primer is demonstrated again by the fact that from 4 to 6 times more activity was found in cyclopentenyl fatty acids after incubation with [l-14C] aleprolic acid, then with [l-14C] acetate. Chain-lengthening as a mechanism for the biosynthesis of cyclopentenyl fatty acids is borne out by the results obtained by Cl-decarboxylation via Schmidt-degradation of individual fatty acids (Table II). On one hand, after incubation of seeds with [l-14C] aleprolic acid, almost no activity is found in Cl of the cyclopentenyl fatty acids; indeed the label should be located solely in the carbon atom next to the cyclopentene ring. On the other hand, figures obtained after incubation with [l-14C] acetate allow one to conclude that an inactive C6 -unit (aleprolic acid) is lengthened to hydnocarpic acid (Cl,) by exactly five active C2-units, as shown by the 20.4% for Cl-activity in this acid. The high values for Cl-activity in chaulmoogric and hormelic acids, the higher homologs of hydnocarpic acid, indicate elongation by separate enzyme systems of preformed hydnocarpic acid. The pattern of Cl-label in straight-chain fatty acids corresponds to that of cyclic acids. Discussion Aleprolic acid has been established as a precursor for cyclopentenyl fatty acids in tissues of Flacourtiaceae species. In contrast to C. uuZgaris [8], higher plants other than Flacourtiaceae could not use this compound as a precursor. Discrimination against aleprolic acid may occur at the acyl-CoA synthetase level or at the level of enzymes mediating the transfer of acyl chains from CoA to acyl-carrier protein, as has been discussed earlier [S]. The occurrence of the non-proteinogenic amino acid cyclopentenylglycine in tissues of Flacourtiaceae (ref. 16, and Cramer, U. and Spener, F., unpublished) suggests that this amino


acid may serve as a precursor of aleprolic acid via transamination and oxidative decarboxylation. In seeds of Flacourtiaceae, mainly straight-chain fatty acids were found labelled after incubation with [l-14C] acetate, whereas aleprolic acid was incorporated almost exclusively into cyclic fatty acids. This may reflect the actual availability of added precursors for either straight-chain and cyclic fatty acid biosynthesis. On the other hand, compartmentation and/or two distinct synthesizing systems for straight-chain and cyclic fatty acids, respectively, are conceivable as well. In either system, however, C 16-fatty acids were synthesized de novo and then elongated. In higher plants, the elongation of palmitate to higher homologs by special “elongases” has been observed by Jaworski et al. [17] and other workers, as summarized by Harwood [18]. Acknowledgements This study was supported by a grant from the Deutsche Forschungsgemeinschaft. We are also indebted to Ir. J.J. Bos of the Landbouwhogeschool, Wageningen, and to Mr. P.R. Weissich of Honolulu Botanic Gardens for providing us with plant material. References 1 Hegnauer. R. (1966) in Chemotaxonomie der Pflanzen, Vol. 4. PP. 155-168. BirkhBuser-Verlag. Base1 2 Spener, F. and Mangold, ILK. (19’74) Biochemistry 13, 2241-2248 3 Spener. F.. Staba, E.J. and Mangold, H.K. (1974) Chem. Phys. Lipid 12. 344-350 4 Spener. F. and Mangold, H.K. (1975) Phytochemistry 14, 1369-1373 5 Van Dorp. D.A., Beerthuis. R.K., Nugteren, D.H. and Vonkeman. H. (1964) Biochim. Biophys. Acta 90. 204-207 6 Bergstrijm. S.. DanieLwon. H. and Samuehson. B. (1964) Biochim. Biophys. Acta, 90, 207-210 7 Hamberg, M.. Svensson, 1.. Wakabayashi. T. and Samuelsson, B. (1974) Proc. Natl. Acad. Sci. U.S. 71, 345-349 8 Spener, F. (1975) Eur. J. Biochem. 53. 161-167 9 Yano, I., Morris, L.J.. Nichols, B.W. and James, A.T. (1972) Lipids 7. 35-45 10 Nichols, B.W. (1964) in New Biochemical Separations (James. A.T. and Morris, L.J.. eds.), pp. 321337, Van Nostrand, London 11 Kannangara. C.G. and Stumpf, P.K. (1972) Arch. Biochem. Biophys. 148,414-424 12 Kannangara, C.G. and Stumpf, P.K. (1972) Arch. Biochem. Biophys. 152, 83-91 13 Bruinsma, J. (1961) Biochim. Biophys. Acta 52. 576-578 14 Appleby. R.S.. Gurr, M.I. and Nichols. B.W. (1974) Eur. J. Biochem. 48, 209-216 15 Aronsson. P. and Gtirtler, J. (1971) Biochim. Biophys. Acta 248. 21-23 16 Spener, F. and Dieckhoff. M. (1973) J. Chromatogr. Sci. 11, 661-662 17 Jaworski. J.G.. Goldschmidt. E.E. and Stumpf. P.K. (1974) Arch. Biochem. Biophys. 163. 769-776 18 Harwood, J.L. (1975) in Recent Advances in the Chemistry and Biochemistry of Plant Lipids (Galhard, T. and Mercer, E.I.. eds.), pp. 43-93, Acad. Press, London