Carotenoid Biosynthesis Pathway: From Acetyl-CoA to Mevalonic Acid and Beta-Carotene
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Carotenoid biosynthesis follows a universal pathway shared by higher plants, algae, bacteria, and fungi, proceeding through the common precursors of all terpenoid compounds — mevalonic acid and isopentenyl pyrophosphate. Research into carotenoid biosynthesis established this shared route across these very different groups of organisms, which is why findings in one system often illuminate the others.
Carotenoid biosynthesis can be divided into several stages. The first is the formation of mevalonic acid from acetyl coenzyme A (Fig. 1). The sequence of reactions leading from acetyl-CoA to mevalonic acid is well studied in animal organisms. As noted earlier, animals cannot synthesize carotenoids themselves, yet they do synthesize mevalonic acid, which serves as a precursor in the synthesis of other compounds, in particular sterols.
The plant enzymes involved in the synthesis of β-hydroxy-β-methylglutaryl-CoA (HMG-CoA) — acetoacetyl-CoA thiolase and HMG-CoA synthase — have not been investigated to date. There are, however, reports that the chloroplasts of higher plants, as well as the chromoplasts of mature fruits of annual pepper, contain the enzyme HMG-CoA reductase, which is able to convert HMG-CoA into mevalonic acid.
How is mevalonic acid formed from acetyl-CoA?
Mevalonic acid is formed directly from intermediates of the reductive pentose phosphate cycle, as demonstrated by K. Grumbach and B. Forn in a series of revealing experiments. They incubated functionally active spinach plastids with several substrates: CO2, acetic, pyruvic, phosphoenolpyruvic, 3-phosphoglyceric, and mevalonic acids. All of these were taken up by the plastids, and all except phosphoenolpyruvic acid were incorporated into β-carotene.
Although the exact route by which acetyl-CoA forms from photosynthetically fixed carbon dioxide has not been clarified, kinetic analysis of substrate incorporation showed that acetyl-CoA is formed directly from intermediates of the reductive pentose phosphate cycle — from phosphoglyceric and pyruvic acids as precursors — and is then used either for fatty acid synthesis or for the formation of mevalonic acid. 
Such studies have not been carried out on isolated chromoplasts. It should be noted, however, that the chromoplasts of yellow daffodil flowers, which are able to synthesize carotenoids from isopentenyl pyrophosphate, contain all the enzymes needed to convert carbon dioxide, acetic acid, 3-phosphoglycerate, and so on into acylated lipids.
These results, together with the finding that isolated chromoplasts of annual pepper can convert HMG-CoA into mevalonic acid, support the conclusion that the first stage of carotenoid synthesis is the formation of mevalonic acid from acetyl-CoA, as shown in Fig. 1. 
The possibility that mevalonic acid synthesized in the cell cytoplasm enters the chromoplasts must also be considered. During the transformation of annual pepper chloroplasts into chromoplasts, the membranes of the plastid envelopes acquire the ability to let this substance pass through.
This permeability gradually increases, reaching a maximum when the ratio of carotenoids to chlorophyll equals 1.66, after which some decline in membrane permeability to mevalonic acid is observed. Even in fully formed chromoplasts, however, it remains at a fairly high level.
How is mevalonic acid converted into isopentenyl pyrophosphate?
The next stage in carotenoid synthesis is the conversion of mevalonic acid into isopentenyl pyrophosphate (Fig. 2). The first reaction involves the enzyme mevalonate kinase. The conversion of mevalonyl-5-phosphate into mevalonyl-5-pyrophosphate was carried out in the presence of mevalonyl-5-phosphokinase. The presence of these enzymes and the reactions they catalyze has been recorded for many plants and microorganisms.
These enzymes were also demonstrated in experiments with isolated chromoplasts of annual pepper. Mevalonate-5-pyrophosphoanhydrase, which participates in the reactions forming isopentenyl pyrophosphate, has been purified from yeast and from the latex of the rubber tree (Hevea). Isopentenyl pyrophosphate itself was detected in all the objects studied. Moreover, it was shown to be converted into carotenoids in preparations of plant chloroplasts and chromoplasts.
From isopentenyl pyrophosphate to geranylgeranyl pyrophosphate
At the stage of converting isopentenyl pyrophosphate into geranylgeranyl pyrophosphate with the subsequent formation of phytoene, isopentenyl pyrophosphate (IPP) is isomerized into dimethylallyl pyrophosphate (DMAPP). This is followed by prenyltransferase reactions that proceed via geranyl and farnesyl pyrophosphates to geranylgeranyl pyrophosphate.
Evidence for the presence of isopentenyl pyrophosphate isomerase (IPP isomerase) in chromoplasts was obtained from studies of tomato plastids, and two isoenzymes of IPP isomerase were found in pumpkin fruit. The literature contains no data on the incorporation of DMAPP into carotenoids; however, the inhibition of the conversion of mevalonic acid into carotenoids by iodoacetamide indicates that DMAPP is an intermediate in carotenoid synthesis. 
The ability of geranyl pyrophosphate to be converted into carotenoids was demonstrated in preparations of many higher plants, including the plastids of carrot roots and tomato fruits. Free geraniol was also incorporated into β-carotene by carrot root discs, apparently because of the presence of enzymes able to phosphorylate geraniol.
Plastid systems of carrot roots and tomato fruits were also shown to be able to incorporate farnesyl pyrophosphate into β-carotene. Subsequently, two molecules of geranylgeranyl pyrophosphate (GGPP) combine to form prephytoene pyrophosphate, whose dephosphorylation leads to the first C40 carotenoid (Fig. 3).
Why is cis-phytoene the first carotenoid synthesized?
Phytoene is the first C40 compound synthesized in chromoplasts, as experiments incorporating radioactive isopentenyl pyrophosphate into isolated annual pepper chromoplasts have shown. Similar results were obtained when daffodil chromoplasts were studied. In isolated annual pepper chromoplasts, phytoene is present as trans (0.2 %) and cis (99.1 %) isomers.
Experiments performed with annual pepper fruits in vivo showed that a noticeable amount of labeled mevalonic acid is incorporated only into cis-phytoene. These data clearly show that cis-phytoene is the first carotenoid synthesized in chromoplasts. In chromoplasts, as in higher plant tissues generally, the first C40 compound is the cis isomer — although in some microorganisms the first carotenoid synthesized is the trans isomer of phytoene.
A protein complex able to convert isopentenyl pyrophosphate into phytoene was isolated from acetone powders of tomato plastids. This complex was purified by ammonium sulfate fractionation and on a Biogel A column without loss of its constituent activities (IPP isomerase, prenyltransferase, phytoene synthetase). Its molecular weight is 166,000 or 200,000.
The complex is active in the presence of manganese ions, and its activity increased 6–7-fold in the presence of ATP. The role of ATP in stimulating activity is unclear, since ATP is not directly incorporated into the reactions occurring during the conversion of isopentenyl pyrophosphate into phytoene. ATP may participate in stabilizing the complex or act as an allosteric regulator of enzyme activity.
The phytoene synthetase complex
The phytoene synthetase complex is highly unstable. Under conventional ionic protein purification methods on DEAE-cellulose it completely lost its ability to synthesize phytoene, although IPP isomerase and prenyltransferase activities were retained. Moreover, during fractionation on DEAE-cellulose the complex dissociated into several components.
To purify IPP isomerase 245-fold from acetone powders of tomato plastids, a multistage procedure was used with sequential ammonium sulfate fractionation, gel filtration on Biogel A 1.5 M, ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephadex G-100, and chromatofocusing.
The purified enzyme was stable for several weeks in 0.1 M potassium phosphate buffer, pH 7.0, containing 2 mM dithiothreitol. In its properties, the IPP isomerase of tomato fruit plastids resembles IPP isomerases from other sources (yeast, pig liver, pumpkin fruit). The enzyme is very sensitive to iodoacetamide, being completely inactivated by it at a concentration of 1 mM.
The IPP isomerase of tomato plastids is also inhibited by the carotenoid synthesis intermediates dimethylallyl and geranyl pyrophosphates, whereas farnesyl pyrophosphate had no significant effect on this enzyme's activity. The molecular weight of tomato fruit plastid IPP isomerase is 34,000 Da. This is considerably lower than the corresponding value for the pig liver enzyme (82,500 Da).
Whether a small molecular weight is characteristic of similar enzymes in other plants is unknown, since to date there have been no reports characterizing IPP isomerases from other plant objects. The only cofactor required for the enzyme's function was divalent metal ions (Mn2+ or Mg2+), and in experiments where both elements were present simultaneously, maximum IPP isomerase activity was recorded.
Prenyltransferase of tomato plastids
Divalent metal ions are also required to activate prenyltransferase. The latter, however, was only slightly inhibited by iodoacetamide at 10 mM, whereas full inhibition of IPP isomerase requires ten times less of this reagent. It should be noted that prenyltransferases from other sources are less sensitive than IPP isomerases to sulfhydryl reagents as well.
The molecular weight of tomato fruit plastid prenyltransferase (from Sephadex G-100 chromatography data) is 64,000 Da, somewhat lower than the corresponding value for analogous enzymes from yeast, pig liver, and pea. The plastid prenyltransferase of tomato fruit catalyzed the following condensation reactions: isopentenyl pyrophosphate + dimethylallyl pyrophosphate, isopentenyl pyrophosphate + farnesyl pyrophosphate, isopentenyl pyrophosphate + geranyl pyrophosphate.
These three activities eluted from the column as a single peak. Chromatographic analysis of the products of reactions catalyzed by the prenyltransferase fraction showed that one of the newly formed components behaves like prephytoene alcohol. This suggests that the enzyme catalyzing the formation of prephytoene phosphate is also present in the prenyltransferase fraction.
This conclusion cannot yet be unambiguous, however, because electrophoretic analysis of the prenyltransferase fraction in the presence of sodium dodecyl sulfate revealed considerable heterogeneity of the preparation. Reliable data can be obtained only by working with an absolutely pure enzyme. The phytoene synthetase complex has a molecular weight of 166,000 or 200,000 Da. IPP isomerase and prenyltransferase together account for roughly 100,000 Da.
This leaves a fraction of 66,000–100,000 Da. It is probably responsible for the formation of phytoene. Phytoene is a colorless compound. Its conversion into colored carotenoids is accompanied by an increase in the size of the conjugated polyene chromophore through a series of desaturation reactions, in each of which two hydrogen atoms are removed and a double bond is introduced.
How is colorless phytoene converted into colored carotenoids?
The intermediates in the desaturation chain are phytofluene, ζ-carotene, neurosporene, and the final product lycopene, as established in experiments on chromoplasts, chloroplasts, and higher plant tissues. The desaturation reactions are easily inhibited by many compounds, including the pyridazinone herbicides SAN 6706 and 9789, which promote the accumulation of phytoene.
Because newly synthesized phytoene is a cis isomer while all colored carotenoids have a trans configuration, isomerization must occur during the desaturation of phytoene. Experiments on the chromoplasts of annual pepper and tomato fruits, as well as on other sources, indicate that conversion of the cis isomer into the trans isomer takes place at the level of phytofluene.
Specifically, the chromoplasts of annual pepper and tomato contained phytofluene in both cis and trans conformations, while all subsequent compounds in the phytoene conversion chain produced by desaturation reactions are trans isomers, and trans-phytoene is present only in trace amounts (Fig. 14). Thus, during the conversion of phytoene into lycopene, four desaturation steps occur with the simultaneous introduction of a double bond, plus a single isomerization of the cis isomer into the trans form. 
G. Britton proposes the existence of an enzyme complex forming an integral part of the membrane structure and participating in the desaturation reactions. From acetone powders of tomato plastids, an enzyme system able to carry out the conversion of phytoene into lycopene was isolated and partially purified by ammonium sulfate fractionation.
The constituent components of this system, however, have not yet been clarified; there are only suggestions about the existence of a single isomerase converting the cis isomer of the carotenoid into the trans form. In the tangerine variety of tomato, poly-cis-carotenoids such as prolycopene accumulate instead of trans-carotenes. The cis isomers of phytofluene, ζ-carotene, and neurosporene were identified as probable intermediates in the synthesis of prolycopene.
At the same time, normal red fruits and the fruits of mutant plants with tangerine coloration differ by only a single gene. Evidently the defective gene in the tangerine variety of tomato encodes an isomerase that converts cis-phytofluene into the trans isomer insufficiently efficiently. Under these conditions, desaturation of the cis isomers occurs, producing poly-cis-carotenoids.
This implies a fairly broad specificity of the desaturases involved in carotene conversion, since they introduce double bonds with high efficiency not only into trans but also into cis isomers. The number of desaturases involved in carotenoid desaturation reactions is not entirely clear at present. The conversion of phytoene into phytofluene was shown to depend on NADP, while the reactions leading from phytofluene to lycopene require FAD and Mn2+.
It appears that at least two desaturases participate in the dehydrogenation of carotenes, with a separate enzyme involved in converting phytoene into phytofluene. Isolated annual pepper chromoplasts promote the conversion of phytoene into lycopene without the addition of NADP. At first glance this contradicts the earlier findings on the participation of NADP in carotene desaturation reactions.
It was shown, however, that intact chromoplasts contain fairly high amounts of endogenous NADP (910 picomoles per 1 mg of protein), which does not rule out the participation of NADP in desaturation reactions. There are also suggestions that metal ions and cytochromes take part in electron transfer during desaturation. Further carotenoid synthesis reactions are connected with the cyclization of the terminal groups.
Cyclization of the terminal groups
Cyclic carotenoids with β and ε rings (see the structural formulas of β- and ε-carotene) are widespread in nature, including in the photosynthetic organelles and chromoplasts of higher plants. To determine the points in the chain of biochemical transformations at which carotenoid cyclization occurs, the herbicidal inhibitor 2-(4-chlorophenylthio)triethylammonium chloride and nicotine are used.
When annual pepper fruits were treated with 2-(4-chlorophenylthio)triethylammonium chloride, lycopene and, to a much lesser extent, neurosporene accumulated, while β-carotene and other cyclic carotenoids disappeared — although this herbicide did not produce such an effect in vitro. Nicotine has a similar effect on carotenoid biosynthesis and accumulation both in vivo and in vitro.
To resolve the contradictions caused by the different effects of the reagents in vivo and in vitro, neurosporene and lycopene synthesized by the microorganism Phycomyces blakesleeanus were incubated with annual pepper chromoplasts. Both precursors were transformed into β-carotene, with lycopene being transformed in preference to neurosporene. Continued: Carotenoid synthesis.
