Metabolic Pathway for Î²-Carotene Biosynthesis - American Chemical

Chapter 34

Metabolic Pathway for β-Carotene Biosynthesis Similarities in the Plant and Animal

Downloaded by UNIV OF LIVERPOOL on December 4, 2016 | http://pubs.acs.org Publication Date: December 20, 1993 | doi: 10.1021/bk-1994-0546.ch034

A. M . Gawienowski Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, MA 01003

Over 75 years ago β-carotene was isolated and assayed in the corpus luteum of the bovine ovary. Its high concentration, the close relation ship of steroid biosynthesis to carotenoid biosynthesis, and Porter's isolation of geranylgeranyl diphosphate from pig liver, caused one to question the assumption that only plant tissues can synthesize carotenoids. Subsequently, we reported in 1969 that bovine corpus luteum can synthesize a small amount of β-carotene from acetate. We further reported that the bovine corpus luteum metabolized C labeled β-carotene to vitamin A aldehyde. Our continued research on the bovine corpus luteum led to the isolation of phytoene, neurosporene, and β-zeacarotene, which are intermediates in the PorterLincoln metabolic pathway to α- and β-carotene in plants. There fore, the biosynthesis of β-carotene is quite similar in the plant and in the bovine corpus luteum. 14

Higher plants contain a large number of isoprenoid compounds with a variety of structures and functions. The terpenoid compounds are formed from multiples of the isoprenoid carbon skeleton. They are derived biosynthetically from isopentenyl diphosphate, the compound which was predicted on the basis of chemical structures of a large number of natural products. In higher plants many of these isoprenoid compounds have important roles in the metabolism and development of the plant. The plant growth regulators gibberellins and abscisic acid are isoprenoid compounds as is β-carotene, the widely present carotenoid. As Gray ( i ) stated in his review article, plants have to be able to produce a wide range of isoprenoid compounds in different amounts in different parts of the plant at different stages of growth and development. Since all these compounds are produced by a common biosynthetic pathway, the plant must have excellent control mechanisms to ensure the synthesis of needed compounds at the right place and time (Figure 1).

0097-6156/94/0546-0401$06.00/0 © 1994 American Chemical Society Huang et al.; Food Phytochemicals for Cancer Prevention I ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

402

FOOD PHYTOCHEMICALS I: FRUITS AND VEGETABLES

Acetyl C o A

\ Acetoacetyl C o A HM

J

CoA

\


Cytokinin s i d e c h a i n s ^ Monoterpenes

Mevalonate

I

IPP

\

GPP FPP Phytyl s i d e c h a i n s Gibberellins

Rubber

-GGPP-

I

— •

Sterols

Carotenoids

Ubiqunone sidechains-*— Decaprenyl P P

Figure 1. Synthesis of isoprenoids to various terpenoids.

The pathway for the biosynthesis of isoprenoid compounds in plants is based a great deal on the sterol synthesis pathway studied for many years in animals and yeast. In plants, isoprenoid biosynthesis can be seen as a major path way (Figure 2) from acetyl CoA via mevalonate and isopentenyl diphosphate to long-chain prenyl diphosphates, with a large number of branch points leading to the separate isoprenoid compounds (Figure 3). Formation of Prenyl Diphosphates Isopentenyl diphosphate is the key intermediate in the formation of isoprenoid compounds. For most isoprenoid compounds, polymerization of C5 units (Figure 4) is required to produce longer chain prenyl diphosphates which are the substrates for various enzymes at the branch points leading to the synthesis of the broad spectrum of isoprenoid compounds (Figure 5). According to Gray (1987), labelled isopent enyl diphosphate has been shown to be an excellent precursor of many isoprenoid compounds in extracts of various plants. Carotenoids were reported in the ovary in 1913 (2) and again in 1932 (3). The bovine corpus luteum contains relatively high concentrations of β-carotene, amounting up to 60 μg/g of tissue weight. Retinal (4) has also been isolated from this tissue. Corpus luteum tissue, when sliced and incubated with β-[15,15'- Η] carotene, yielded radioactive retinal (5). This indicated the ovary possesses the enzymes to synthesize retinal in situ, which may have a role in reproductive functions. At ovulation time, specific activity of the carotene cleavage enzymes was two-fold greater in the ovary than in the intestine (6). 3

Huang et al.; Food Phytochemicals for Cancer Prevention I ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

GAWIENOWSKI

Metabolic Pathway for β-Carotene Biosynthesis

CH CH C=0 S-CoA 3

HC

OH

CH

H2C

CH2

3

2

C=0 COOH

C=0 CoA

S-CoA

Acetyl CoA Downloaded by UNIV OF LIVERPOOL on December 4, 2016 | http://pubs.acs.org Publication Date: December 20, 1993 | doi: 10.1021/bk-1994-0546.ch034

3

C=0

Acetoacetyl CoA

S-CoA p-Hydroxyl-p-metylglutary-CoA

H

P

OH OH p P=0 o—P-O—P=0 ÇH OH ATP ADP 2 Ç O' ATP ADP H c ' Ary H C OH H C OH ί H C' ÇH H C' CH H C CH COO" COO" ^ . H

2

2

2

: c ;

2

:c:

Η 2 (

0

X

3

2

3

2

3

H

X

2

0 0

Mevalonate

5-P-Mevalonate

O-PP HC H C' 3

O-PP HC H C ^ 2

2

HC^

5-Pyrophosphate mevalonate

-

C N

CH

2

H C 3

*CH

2

3

Dimethylallyl pyrophosphate

O-PP Geranyl pyrophosphate

Isopentenyl pyrophosphate

Farnesyl pyrophosphate

Squalene

Figure 2. Synthesis of squalene.


403





34. GAWIENOWSKI


Geranyl geranyl diphosphate

x2

Figure 4. Biosynthesis of phytoene.


405


406


15- cis Phytofluene

α-Carotene

β-Carotene

Figure 5. Synthesis of carotenoids.


34. GAWIENOWSKI


407

It had been suggested that vitamin A may be required for efficient steroid hormone production (7-9) and that it plays a role in reproductive processes, e.g., maintenance of pregnancy (10). It is also of interest that |3-carotene- C was synthe sized in vitro in the bovine corpus luteum tissue from sodium [1- C] acetate (12). The retinoids in vertebrates represent an essential class of nutrients needed for the maintenance of differentiated epithelial structures, vision, reproductive functions and health (13). Retinoic acid, for example, has multiple effects on cell growth and differentiation and appears to be organogenic in embryogenesis. The effects of retinoic acid on gene expression are believed to be mediated by a class of nuclear receptors that function as ligand-dependent transcription factors (14). Piziak and Gawienowski (9) found that progesterone-4- C can be metabolized by guinea pig placenta tissue to 20a-hydroxyprogesterone to a signi ficant extent in an atmosphere of 95% oxygen:5% carbon dioxide. Retinol increased this metabolism tenfold. Vitamin A deficiency is known to have an adverse effect on the size of the ovary and on the integrity of the uterus and would thus have an effect on reproductive ability (75). But a more direct effect on steroid metabolism probably exists. One clue that suggests this fact is the disappearance of vitamin A from the ovary after menopause (16). Ganguly et al. discovered that vitamin A deficient rats secreted less than normal amounts of progesterone and 20a-hydroxyprogesterone (77). Juneja et al. (8) reported a decrease in conversion of Δ -3β-hydroxysteroids into A -3-ketosteroids in tissues that were only mildly deficient in vitamin A . 14

14


l4

5

4

Isolation of Phytoene in the Bovine Ovary In order to understand better the role of the carotenoids in the bovine ovary, we analyzed the corpus luteum for phytoene. Phytoene is a colorless 40-carbon compound that serves as a precursor for many carotenoids in plants. Porter isolated geranylgeranyl diphosphate synthetase, which aids in the biosynthesis of geranylgeranyl diphosphate a precursor of phytoene (75), from pig liver (79). The formation of phytoene in plants arises from a head to head conden sation of two geranylgeranyl diphosphate molecules. Whenever β-carotene has been observed, phytoene has usually been found in substantial amounts (78). Phytoene has also been studied in some mammalian tissues (20,21). Phytoene was a logical precursor of β-carotene to be investigated in the bovine ovary with and without a corpus luteum. Since phytoene has been well studied in plants, we were able to adapt the plant analytical procedures to ovarian tissue analysis. Materials and Methods Standard phytoene was donated by Hoffmann-LaRoche, Inc. and M . Mathews-Roth of Harvard University. Most of the experimental procedures were carried out according to those used by Davies (22). The N-bromidesuccinimide method for derivative formation was reported by Zechmeister (23). Ovaries were obtained fresh from a local abattoir. Ovaries with and without their corpora lutea were homogenized and extracted with a mixture of chloroformmethanol (2:1, v:v). The sample was saponified with a 60% (w/v) potassium hydroxide solution, in the dark under nitrogen. The dried sample was dissolved in petroleum ether and applied to a column (1 χ 15 cm) containing 25 g neutral



408


alumina, Brockman activity grade III. The extract was eluted with petroleum ether containing increasing concentrations of diethyl ether. Visible and ultraviolet absorption spectra of the eluted fractions were determined in a Cary Model 14, Beckman Model 24 or Acta M V I spectrophoto meter. Quantitative determinations of phytoene were carried out in known volumes of petroleum ether at 286 nm by their extinction coefficients (Figure 6). After the quantitative analysis, a N-bromosuccinirnide derivative of ζ-carotene was (374, 395, 420 nm) made utilizing the Zechmeister (23) procedure (Figure 6). Phytoene standards were also treated under the same conditions and gave the identical derivative. Iodine catalyzed photoisomerization of the extract was carried out in hexane in quartz spectrophotometer cuvettes (22). Illumination was performed under two parallel fluorescent lamps (65 W) at a distance of 40 cm. The elution pattern for phytoene correlates well with the literature. The phytoene eluted from the column in solvent containing between 2 to 5% diethyl ether in petroleum ether, which agrees with Davies (22) and Than et al. (24). Results A total of five bovine ovaries were analyzed for phytoene and the results are given in Table I. A large cyst, containing 17.3 ml of fluid, was found in ovary number II which also contained the smallest amount of phytoene per gram of tissue. The corpus luteum of ovary number IV was removed before the analysis. As can be noted, it had the second lowest phytoene concentration. Table I. Phytoene Concentration in Bovine Ovaries Ovary: I Weight (g) Phytoene cone, (^g/g)

33.9 8.20

IP

III

IV

V

50.9 0.04

22.2 6.41

10.0 4.22

21.4 10.94

Contained a large cyst

Neurosporene An additional carotenoid was isolated with the Davies (22) procedure. It had an absorption spectrum of 414, 440, 469 and was observed at a concentration of 1.77 μg/g of tissue (Figure 7). On the basis of UV/VIS spectra and elution pattern, its identity was determined to be neurosporene (7,8-dihydro-\{/, ψ-carotene). The cis isomer was converted to the trans form by iodine catalyzed photoisomerization (22). Neurosporene was identified in six bovine corpora lutea by spectra and elution pattern. Discussion The higher plants and mammals contain a wide array of isoprenoid compounds which relate to the active isoprene compound, isopentenyl diphosphate. Just as it


GAWIENOWSKI



34.


409


410


Figure 7. UV-visible spectra of neurosporene standard (-

-) and sample


34. GAWIENOWSKI


has been found in plant biosynthesis of β-carotene via the Porter-Lincoln pathway, these tetraterpenoid precursors are apparently similar in the ovary. With the demonstration of the biosynthesis of acetate- C to β-carotene- C in the corpus luteum tissue, one can suspect that plants and mammals have a similar β-carotene synthesis pathway. The isolation of β-carotene, α-carotene, phytoene, βzeacarotene (25), and neurosporene, from the ovary, are good indicators of a Porter-Lincoln type system in the ovary (Figure 8). The synthesis of retinal- C from β-carotene- C in the ovary indicates the enzymes are there for retinoid formation. Retinal has also been isolated from the corpus luteum. This also relates to the known retinoid action on steroid metabolism. Retinoids have an effect on cell growth, differentiation and embryogenesis. Also, the effects of the retinoid on gene expression are thought to be mediated by a family of nuclear receptors that function as ligand-dependent transcription factors (14). These functions would only require minute quantities of the retinoids. Phytoene was isolated and identified in five bovine ovaries. This plus our past analyses of β-zeacarotene (25), β-carotene, α-carotene, neurosporene, and retinal in the bovine ovary are good indicators of a Porter-Lincoln type metabolic system in the ovary. The metabolic pathway has been well established in plants, but remains virtually unknown in mammals (18). Glycolysis, the Krebs cycle and other metabolic pathways are well recognized in plants as well as in mammalian systems. Therefore, the similarities of the isoprenoid pathway to β-carotene in plants and mammals should aid in the recognition of another related metabolic pathway. 1 4

14


411

370

14

14

410

450

(nm)

Wavelength

Figure 8. UV-visible spectra of β-zeacarotene standard (—) and sample (


).

412


Acknowledgements The author thanks Ν. H. Zawia and J. J. Boniface for technical assistance.


Literature Cited 1. Gray, J. C. Control of Isoprenoid Biosynthesis in Higher Plants; Callow, J. Α., Ed.; Advances in Botanical Res.; Academic Press: New York, 1987; Vol. 14, pp 25-91. 2. Escher, H. Hoppe-Seyler's Z. Physiol. Chem. 1913, 83, 198-211. 3. Kuhn, R.; Lederer, E. Hoppe-Seyler's Z. Physiol. Chem. 1932, 206, 41-64. 4. Austern, B. M . ; Gawienowski, A. M . J. Reprod.Fert.1969, 19, 203-205. 5. Gawienowski, A . M.; Stacewicz-Sapuncakis, M . ; Longley, R. J. Lipid Res. 1974, 15, 375-379. 6. Sklan, D. Internat. J. Vit. Nutr. Res. 1983, 53, 23-26. 7. Grangaud, R.; Nicol, M . ; Desplanques, D. Amer. J. Clin. Nutr. 1969, 22, 9911002. 8. Juneja, H.; Murthy, S.; Ganguly, J. Biochem. J. 1966, 99, 138-145. 9. Piziak, V . K.; Gawienowski, A . M. Comp. Biochem. Physiol. 1972, 42B, 201203. 10. Moore, T. Vitamin A; Elsevier: Amsterdam, 1957; p 329. 11. Thompson, J.; Howell, M . ; Pitt, G. In Agents Affecting Fertility; Austin, C.; Perry, J., Eds; J. & A Churchill Ltd.: London, 1965; pp 32-46. 12. Austern, Β. M.; Gawienowski, A. M . Lipids 1969, 4, 227-229. 13. Goodman, D. S., N. Engl. J. Med. 1984, 310, 1023-1031. 14. Rajan, N . ; Kidd, G.; Talmage, D.; Blaner, W.; Suhara, Α.; Goodman, D. S. J. Lipid Res. 1991, 32, 1195-1204. 15. Truscott, B. L. Anat. Record. 1947, 98, 111-126. 16. Ragins, A. B., Popper, H. Arch. Pathology 1942, 34, 647. 17. Ganguly, J.; Pope, S.; Thompson, J.; Toothill, J.; Edwards-Webb, J.; Waynforth, H. Biochem. J. 1971, 122, 235-239. 18. Britton, G. The Biochemistry of Natural Pigments; Cambridge Univ. Press: New York; 1983, pp 46-66. 19. Porter, J. W.; Nandi, D. L. Arch. Biochem. Biophys. 1964, 105, 7-19. 20. Mathews-Roth, M. M . In Carotenoids as Colorants and Vitamin A Precursors; Bauernfeind, J., Ed.; Academic Press: New York, 1981; pp 755-781. 21. Mathews-Roth, M. M . ; Crean, C.; Clancy, M . Nutr. Reports Int. 1978, 17, 581584. 22. Davies, Β. H . in Chemistry and Biochemistry of Plant Pigments; Goodwin, T., Ed.; Academic Press: New York, 1976; pp 38-155. 23. Zechmeister, L.; Koe, Β. K. J. Am. Chem. Soc. 1954, 76, 2923-2926. 24. Than, Α.; Bramely, P. M . ; Davies, B . H.; Rees, A . F. Phytochem. 1972, 11, 3187-3192. 25. Gawienowski, A . M.; Soderstrom, D. N.; Tan, B . Biotech. Appl. Biochem. 1986, 8, 190-194. RECEIVED October 4, 1993


Metabolic Pathway for Î²-Carotene Biosynthesis - American Chemical

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