21.6: Biosynthesis and Metabolism of Isoprenoids

Structure and Occurrence

In tocopherols, the C16 side chain is saturated, whereas tocotrienols contain three trans double bonds. Together, these two groups are collectively called tocochromanols. In essence, the tocopherols have a 20-carbon phytyl tail (including the pyranol ring) and four chiral centers in total, with variable numbers of methyl groups attached to the benzene ring, while the tocotrienols have a 20-carbon geranylgeranyl tail with double bonds at the 3', 7' and 11' positions relative to the ring system. Tocopherols contain three chiral carbons, one at C2 in the chromanol ring and two in the side chain at C4′ and C8′ with R,R,R stereochemistry. The four main constituents of the two classes are termed alpha (5,7,8-trimethyl), beta (5,8-dimethyl), gamma (7,8-dimethyl), and delta (8-methyl). In contrast to the tocopherols, the tocotrienols have only one chiral center. Plastochromanol-8 is an analog of γ-tocotrienol with a much longer side chain. Their structures are shown in Figure \(\PageIndex{1}\).

Plants and other oxygenic photosynthetic organisms, such as algae and some cyanobacteria, synthesize tocochromanols, but they are essential components of animal diets. Of these, only natural R,R,R-α-tocopherol is now designated ‘vitamin E’, as explained below. However, the other tocopherols are sometimes termed ‘vitamers’ (some claim incorrectly - nor should all forms be termed isomers strictly speaking). In the United States, the current recommended dietary allowance (RDA) for adults is 15 mg of α-tocopherol daily. In plants, tocochromanol content and composition vary widely, and photosynthetic plant tissues contain 10-50 μg of tocochromanols per gram of fresh weight. α‑Tocopherol is present only in photosynthetic membranes of plant leaves, while γ-tocopherol and other forms are found principally in fruits, seeds, and nuts. Tocopherols are present in all photosynthetic organisms, but tocotrienols occur only in certain plant families.

Seed oils are a significant source in the human diet, and Table 1 lists the tocopherol compositions of some unrefined oils. Sunflower and olive oils are good sources of α‑tocopherol, and palm oil is a good source of the tocotrienols. In general, tocotrienols are abundant only in seeds and fruits, especially those of monocots such as wheat, rice, and barley; however, palm oil is a major commercial source. In leaf tissue, α-tocopherol is often the main form, while γ-tocopherol is the primary tocopherol of many seeds. Plastochromanol-8 was first identified in the leaves of the rubber tree (Hevea brasiliensis) but has since been found in many other plants, including rapeseed and maize, typically at lower levels than the tocopherols. Plant tissues also contain tocopherol esters of fatty acids, which may serve as a storage form.  Unesterified tocopherols are not released during digestion in animals, so they may not contribute to vitamin E activity.

An unusual tocopherol, termed marine-derived α-tocomonoenol, occurs alongside α-tocopherol in a wide range of marine fish species and appears to scavenge free radicals more efficiently at low temperatures. A related isomer with a Δ11 double bond has been found in palm oil and kiwi fruit. While pumpkin seeds contain both α- and γ-tocomonoenols, other plant species contain β-, γ-, and δ-tocomonoenols with unsaturation in the terminal isoprene unit of their side chains. Tocochromenols, or 3,4-dehydrotocopherols, i.e., with a double bond in the pyranol ring, are also known, in addition to more complex tocopherol-like molecules. The structures of tocomonoenols are shown in Figure \(\PageIndex{2}\).

α-Tocopherol is a minor but ubiquitous component of the lipid constituents of animal cell membranes (non-raft domains), with estimates ranging from 1 tocopherol molecule to 100 to 1000 phospholipid molecules, depending on the membrane. The hydrophobic tail lies within the membrane, as expected, and the polar head group faces the surface but sits below the phospholipid phosphate moieties. Hydrogen bonding between the hydroxyl groups and the phosphate may be limited, depending on the membrane's hydration. In contrast, α-tocopherol has a strong affinity for polyunsaturated fatty acids, where the chromanol unit may interact with the double bonds, suggesting that tocopherol is located deep within the membrane.

α-Tocopheryl phosphate has recently been detected at low levels in plasma, liver, and adipose tissue. Its structure is shown in Figure \(\PageIndex{3}\). Together with catabolic tocopherol metabolites, it has important biological properties.

During vegetable oil refining, much of the natural tocopherols are lost or destroyed. Most commercial vitamin E is therefore prepared by chemical synthesis from trimethylhydroquinone and phytyl bromide. The resulting product is a mixture of eight stereoisomers (from R,R,R- to S,S,S-methyl groups) of α-tocopherol, with the various stereoisomers differing by a factor of two in biological activity, as a consequence of the stereochemistry of position 2 in the chromanol ring (i.e., 2S-α- compared to 2R-α-tocopherol). It is usually administered in vivo as the acetate derivative. Tocopherols are not traditionally regarded as effective antioxidants in polyunsaturated seed oils. At higher concentrations, they can even act as pro-oxidants, although the reasons for this are not well understood.

Biosynthesis and Functions of Tocochromanols in Plants

The mechanism of tocopherol biosynthesis has been elucidated and involves the coupling of phytyl diphosphate with homogentisic acid (2,5-dihydroxyphenylacetic acid), followed by cyclization and methylation reactions. Biosynthesis occurs in the plant chloroplast, and most enzymes are located on the inner membrane of the chloroplast envelope. However, growing evidence suggests that plastoglobules associated with the thylakoid membrane may also be involved in this process. The pathway for biosynthesis of tocopherols is shown in Figure \(\PageIndex{4}\).

The aromatic amino acid tyrosine is the basic precursor and is oxidized to p-hydroxypyruvic acid, which, in the first committed step, the enzyme p-hydroxyphenylpyruvate dioxygenase converts to homogentisic acid. Homogentisic acid condenses with phytyl diphosphate, derived from phytol obtained by hydrolysis of chlorophyll, in a reaction catalyzed by a prenyl transferase to yield 2-methyl-6-phytyl-plastoquinol, which is first methylated to form 2,3-dimethyl-5-phytyl-1,4-benzoquinol and then converted by the enzyme tocopherol cyclase to γ-tocopherol. A further methylation reaction produces α-tocopherol, while modifications to the pathway produce β- and δ-tocopherols, as well as plastoquinones, which ultimately lead to plastochromanol-8. Tocotrienols and tocomonoenols result from a similar series of reactions but with geranylgeranyl diphosphate and tetrahydrogeranylgeraniol diphosphate, respectively, as substrates in the condensation step. Isoprenoid precursors are also synthesized in the plastid by the non-mevalonate, or 'MEP', pathway.

In plants, tocopherols are found mainly in the chloroplasts of green tissues, but also in seeds, fruits, roots, and tubers. They are especially important as antioxidants, limiting damage from photosynthesis-derived reactive oxygen species under oxidative stress, including high-intensity light stress. The mechanisms for this antioxidant activity are discussed below. However, recent studies suggest that they are just one of several components involved in photoprotection. Certainly, any tocochromanol peroxy radicals formed must be converted back to the original compounds by the concerted action of other plant antioxidants, for example, by ascorbate, glutathione, ubiquinol, or lipoic acid, and antioxidant enzymes, including superoxide dismutase, catalase, and peroxidases. Tocopherols are essential for controlling non-enzymatic lipid peroxidation during seed dormancy and seedling germination. In their absence, malondialdehyde and phytoprostanes increase, and plant defense responses may activate inappropriately.

Evidence suggests that tocopherols play a role in intracellular signaling in plants, regulating jasmonic acid levels in leaves by modulating lipid peroxidation and gene expression, thereby influencing plant development and stress responses. Thus, by controlling the degree of chloroplast lipid peroxidation (redox regulation), they limit the accumulation of lipid hydroperoxides required for jasmonic acid synthesis, which, in turn, regulates the expression of genes that respond to various abiotic stress conditions, including drought, salinity, and extreme temperatures. Tocopherols regulate enzyme translocation to the plasma membrane, possibly by modulating protein-membrane interactions, altering membrane microdomains (lipid rafts), or competing for common binding sites within lipid transport proteins. Additionally, tocopherols are essential for cell wall development in phloem transfer cells under cold conditions. α- and γ‑tocopherol and the tocotrienols may each have distinct functions. For example, γ-tocopherol is reportedly more potent than α-tocopherol in protecting plants from the harmful effects of osmotic stress and is important for seed longevity. Efforts are underway to increase tocopherol levels in plants through selective breeding and genetic manipulation to produce crops with enhanced health benefits for consumers and, potentially, for the plants themselves.

Tocopherols Metabolism in Animals

In animals, the first step in tocopherol digestion is dissolution with other lipids in mixed micelles in the intestines. All tocopherol forms are absorbed to a similar extent in the intestines via transporters in the enterocyte apical membrane with broad specificity for hydrophobic molecules, such as cholesterol, vitamin D, and carotenoids. These include scavenger receptor class B type I (SR-BI), CD36, and NPC1-like intracellular cholesterol transporter 1 (NPC1L1). However, some passive diffusion cannot be ruled out. Transport across the enterocyte may involve cytoplasmic transporters or clathrin-coated vesicles before the tocopherols are incorporated into chylomicrons in free form in the Golgi apparatus for release into the lymph. At the liver, α-tocopherol specifically is taken up from the chylomicrons by a receptor-mediated mechanism with the aid of a specific tocopherol-binding protein (the α-tocopherol transfer protein (α-TTP)), i.e., a 30,500 Da cytosolic protein that has a marked affinity for α-tocopherol and can enhance its transfer between membranes. This recognizes α-tocopherol by the three methyl groups and the hydroxyl on the chromanol ring, as well as by the structure and orientation of the phytyl side chain. It is the chief regulator of whole-body α-tocopherol status and is expressed primarily in the cytosol of hepatocytes in the liver, but has also been reported in other tissues, such as the placenta, at much lower concentrations.

α-TTP ultimately regulates the egress of α-tocopherol selectively from hepatocytes with the aid of the ATP-binding cassette proteins ABCA1 and ABCG for conveyance in the plasma lipoproteins, mainly in the very-low-density lipoproteins or VLDL (and thence to LDL) and HDL in humans, to the peripheral tissues (together with much smaller amounts of γ-tocopherol). Most of the other tocopherol forms are directed toward catabolism. Once in circulation, tocopherol can exchange spontaneously between membranes and lipoproteins, and no specific plasma transport protein for vitamin E has yet been described. Tocopherols transfer from VLDL to peripheral tissues via lipoprotein lipase hydrolysis of triacylglycerols, whereas LDL delivers them via the LDL receptor-mediated uptake pathway. Within peripheral tissue cells, including the central nervous system, α-TTP transports α-tocopherol to wherever it is required in membranes. This process appears to be aided by phosphatidylinositol metabolites. In the brain, apoE-rich lipoproteins transport tocopherol. Tocopherol concentrations vary appreciably among tissues, with the highest in adipose tissue and adrenals, lower in the kidney, heart, and liver, and lowest in erythrocytes.

The "α-tocopherol salvage pathway" is partly due to this process and partly to selective oxidation (see below), resulting in a 20- to 30-fold enrichment of α-tocopherol in plasma (average concentration: 22-28 μM) relative to the other tocopherols. Thus, the process of conserving a specific tocopherol appears to determine the relative vitamin E activities of tocopherols and tocotrienols in vivo rather than their antioxidant potencies, as measured in vitro using model systems. Only α‑tocopherol (including synthetic material) or natural mixtures containing this can be sold under the label 'Vitamin E'. γ‑Tocopherol is the second most abundant form in plasma, and it is present in relatively greater proportions in the skin, adipose tissue, and skeletal muscle, where it has some specific biological properties that are distinct from those of α-tocopherol. Although tocotrienols are more potent antioxidants in vitro, they are not typically detected in tissues; however, they are believed to have some important functions.

Catabolism: The unwanted surplus of tocochromanols other than α-tocopherol may be excreted in the urine and feces in the form of carboxy-chromanols, including the so-called 'Simon metabolites' - tocopheronic acids (carboxyethylhydroxychromans, CEHC) and tocopheronolactones, after oxidative cleavage of much of the phytyl tail, although these are normally detected in the form of conjugates as sulfate or glucuronidate esters, the forms in which they are excreted in feces and urine. For example, for illustrative purposes in liver cells, the first step in the catabolism of γ-tocopherol is ω-hydroxylation by cytochrome P450 (CYP4F2) at the 13' carbon to form γ-13'-hydroxychromanol in the endoplasmic reticulum, followed by ω-oxidation in the peroxisomes to produce γ‑13'‑carboxychromanol, and finally by stepwise β‑oxidation in the mitochondria to cut off two or three carbon moieties from the phytyl chain in each cycle. These steps are shown in Figure \(\PageIndex{5}\).

Researchers have identified various carboxychromanol intermediates for all tocopherols, as well as sulfated forms of the hydroxyl group, in human cell cultures in vitro. Sulfated carboxychromanols are the primary tocopherol metabolites in rodent plasma. Because vitamin E ω-hydroxylase has a high affinity for tocopherols other than the α-form and does not attack those bound to the α-tocopherol transfer protein, this further increases the α‑tocopherol concentration in plasma relative to the others. Some of these catabolic metabolites may have some biological activity in their own right. For example, carboxyethylhydroxychromans derived from γ-tocopherol have been reported to induce apoptosis in cancer cells and exhibit anti-inflammatory effects by inhibiting cyclooxygenases and 5-lipoxygenase (see below). Tocotrienols are catabolized similarly, but with additional steps in which the double bonds are reduced before oxidation; the final carboxyethylchromanols are the same as for tocopherols.

Tocopherols as Antioxidants

Although the syndrome associated with a lack of vitamin E in animal diets has been known for decades, the mode of action and specific location of tocopherols in cell membranes remain unclear. Several theories have been proposed to explain vitamin E's functions in animal cells. In vitro studies have long suggested that a major role is to act as an antioxidant, inhibiting, reducing, delaying, or preventing oxidative damage to unsaturated lipids or other membrane constituents and thereby protecting tissues by scavenging free radicals. For example, vitamin E administration can prevent lipid peroxidation and hepatotoxicity upon exposure to the free radical-generating agent carbon tetrachloride. Lipid peroxidation also causes ferroptosis, an iron-dependent form of nonapoptotic cell death. However, tocopherols have functions beyond antioxidant activity. In non-biological systems, such as food, cosmetics, and pharmaceutical preparations, tocopherols are invaluable antioxidants.

Because of their lipophilic nature, tocopherols are found in membranes or associated with storage lipids, where they are readily available to interact with lipid hydroperoxides. In brief, Reactive Oxygen Species (ROS), of which innumerable forms exist, can be derived by enzymatic or non-enzymatic means and produce superoxide anions and other peroxyl radicals. Superoxide radicals (O₂^•-) ultimately generate highly toxic hydroxyl (^•OH) or alkoxyl radicals, which can abstract a hydrogen atom from bis-allylic methylene groups of polyunsaturated fatty acids under aerobic conditions in vivo in animals and plants to generate lipid peroxyl radicals (LOO^•) and hydroperoxy fatty acids. Singlet oxygen (¹O₂ or O=O) is an especially important ROS (non-radical) in plant photosynthetic tissues. As the radical generation is not enzymatic, all methylene groups between two cis double bonds can potentially participate in the reaction, although not necessarily to the same extent. Tocopherols react rapidly in a non-enzymatic manner, unlike many other cellular antioxidants, which are dependent on enzymes, to scavenge lipid peroxyl radicals, i.e., the chain-carrying species that propagate lipid peroxidation. In model systems in vitro, all the tocopherols (α > γ > β > δ) and tocotrienols are good antioxidants, with the tocotrienols being the most potent.

In general, lipid oxidation proceeds via a chain process mediated by such free radicals, in which the lipid peroxyl radical serves as a chain carrier. In the initial step of chain propagation, a hydrogen atom is abstracted from the target lipid by the peroxyl radical, as shown in Figure \(\PageIndex{6}\).

The main function of α-tocopherol is to scavenge the lipid peroxyl radical before it can react with the lipid substrate, as shown in Figure \(\PageIndex{7}\).

An antioxidant's potency depends on the relative rates of reactions (1) and (2). When a tocopheroxyl radical forms, delocalization of the unpaired electron around the fully substituted chromanol ring system stabilizes it, making it relatively unreactive and preventing propagation of the chain reaction. This also explains the high first-order rate constant for hydrogen transfer from α-tocopherol to peroxyl radicals, as studies of the relative rates of chain propagation to chain inhibition by α-tocopherol in model systems have shown that α-tocopherol scavenges peroxyl radicals much more rapidly than peroxyl radicals react with a lipid substrate.

In biological systems, oxidant radicals can originate from various sources, including singlet oxygen, alkoxyl radicals, superoxide, peroxynitrite, nitrogen dioxide, and ozone. α-Tocopherol is most efficient at protecting against peroxyl radicals in a membrane environment.

The reaction of the tocopheroxyl radical with a lipid peroxyl radical, as illustrated, yields 8α-substituted tocopherones, which are readily hydrolyzed to 8α-hydroxy tocopherones that rearrange spontaneously to form α-tocopherol quinones. In an alternative pathway, the tocopheroxyl radical reacts with the lipid peroxyl radical to form epoxy-8α-hydroperoxytocopherones, which hydrolyze and rearrange to epoxyquinones. Tocopherol dimers and trimers may also be formed as minor products. These reactions are shown in Figure \(\PageIndex{8}\).

Free radical-mediated lipid peroxidation is the primary pathway of lipid oxidation in humans, and α-tocopherol is a key antioxidant. Still, it does not efficiently scavenge the nitrogen dioxide radical, the carbonate anion radical, or hypochlorite. Vitamin E forms with an unsubstituted 5-position, such as γ-tocopherol, are an exception to the rule that tocopherols have similar antioxidant properties, in that they can trap electrophiles, including Reactive Nitrogen Species (RNS), which increase during inflammation. The enzyme nitric oxide synthase continuously produces large amounts of nitric oxide (NO•), which can react with superoxide to form peroxynitrite (ONOO^-). This potent and versatile oxidant can attack a wide range of biological targets. It induces lipid peroxidation and nitrates aromatic compounds and unsaturated fatty acids, while isomerizing cis-double bonds in fatty acids to the trans configuration. γ-Tocopherol is superior to α‑tocopherol in detoxifying the NO₂ radical and peroxynitrite with formation of 5-nitro-γ-tocopherol, as shown in Figure \(\PageIndex{9}\) below.

This occurs in vivo, and 5-nitro-γ-tocopherol concentrations are elevated in the plasma of subjects with coronary heart disease and in carotid-artery atherosclerotic plaque.

In plant and animal tissues, tocopherols can be regenerated from tocopheroxyl radicals via a redox cycle mediated by various endogenous antioxidants, including vitamins A and C and coenzyme Q, thereby significantly extending their biological potency. Vitamin C (ascorbic acid) may be especially important in aqueous systems. However, it may also act at the membrane surface to regenerate α-tocopherol, while being oxidized in turn to dehydroascorbic acid. This can be regenerated to its reduced form by glutathione (GSH), producing glutathione disulfide (GSSG), which can subsequently be enzymatically reduced by glutathione reductase using NAD(P)H as a cofactor. In plants, an NAD(P)H-dependent quinone oxidoreductase is involved early in regeneration. In contrast, tocopherol cyclase, an enzyme involved in tocopherol biosynthesis, reintroduces the chromanol ring. These linked cycles, known as the antioxidant network, are illustrated in Figure \(\PageIndex{10}\).

Thus, tocopherols are only one component of a complex web of metabolites and enzymes in tissues that have antioxidant activities and act by various mechanisms, including stimulating genes involved in signaling responses to environmental stresses. One antioxidant mechanism involves enzymes such as superoxide dismutase, catalase, and glutathione peroxidase, which remove free radicals and reactive species. Additionally, electron donors such as glutathione, tocopherols, ascorbic acid, vitamin K, coenzyme Q, and thioredoxin also scavenge free radicals. Metal-binding proteins, such as transferrin, metallothionein, haptoglobin, and ceruloplasmin, exhibit antioxidant activity by sequestering pro-oxidant metal ions, including iron and copper. However, some metals, such as selenium and zinc, exhibit antioxidant properties. Other antioxidants, including flavonoids, carotenoids, phenolic acids, and tocopherols, enter animal tissues through the food chain. Although this discussion has focused on effects on lipids, free radicals can damage proteins, DNA, and, indeed, virtually any native substance in living organisms.

Biological Functions of Tocochromanols in Animals

Vitamin E deficiency has been detected in patients with fat malabsorption, cystic fibrosis, Crohn's disease, liver disease, pancreatic insufficiency, and premature infants. Vitamin E deficiency impairs normal immune function in animals and humans, and vitamin E repletion can correct it. It also displays activity against nonalcoholic hepatosteatosis. Although various proposals for the pathogenic mechanism exist, none has been generally accepted. After the discovery of vitamin E's effects on fertility in laboratory animal studies, researchers documented its importance in the development of various tissues and organs, including the brain and nerves, muscles and bones, skin, bone marrow, and blood; most of these effects are specific to α-tocopherol. However, no evidence supports a vitamin E effect on fertility in humans, as was initially observed in rats. The rare genetic disorder “Ataxia with Isolated Vitamin E Deficiency,” or “AVED,” results from mutations in the gene coding for α-TTP. It causes death of cerebellar Purkinje cells, but α-tocopherol administration prevents this and the subsequent development of clinical symptoms.

Tocopherols appear to inhibit many inflammation-associated enzymes in vitro and in animal models and may contribute to the amelioration and treatment of some chronic diseases. However, data on the effects of vitamin E on in vivo biomarkers of oxidative stress are inconsistent. Oxidized vitamin E metabolites, i.e., those that have reacted as antioxidants, are barely detectable in tissues, and vitamin E maintenance in vivo does not appear to be associated with its regeneration. Significant differences appear between results from studies in laboratory animals and those in humans. Thus, clinical intervention studies have not confirmed suggestions that dietary vitamin E supplementation may reduce lipid oxidation in low-density lipoproteins in humans, as they have not shown reduced incidence or severity of atherosclerosis. However, some conditions have been linked to benefits. Indeed, some suggest that excessive vitamin E supplementation may even be harmful. One study suggested that relatively high doses of natural α-tocopherol over a long period are required to show a significant reduction in urinary F2-isoprostanes, considered the most reliable in vivo marker of oxidative stress. While many fat-soluble antioxidants are present in the diet, only α-tocopherol is a vitamin. Some studies suggest that a network of cellular antioxidant defenses may protect tocopherol from functioning as an antioxidant in some tissues in vivo, so tocopherol is used only when other antioxidants are depleted. However, no experimental evidence supports this hypothesis.

At the cellular level,  RRR-α-tocopherol has been shown to inhibit protein kinase C. In the process, it inhibits the assembly and radical-producing activity of NADPH oxidase in monocytes. Similarly, vitamin E suppresses xanthine oxidase expression in the liver, a source of reactive oxygen species. Thus, α-tocopherol may reduce free radical levels by preventing their production rather than scavenging them. Its physical presence in membranes adjacent to polyunsaturated fatty acids may thus limit autoxidation.

With the discovery that the antioxidant effects of various tocopherols and tocotrienols have little relation to their vitamin E activities in vivo, we have realized they have other functions in tissues, most of which are specific to α-tocopherol. Most current research focuses on how vitamin E and its metabolites function in signaling and, controversially, in regulating gene activity. While most other vitamins are essential cofactors for specific enzymes or transcription factors, researchers have yet to identify a receptor that binds specifically to vitamin E. By preventing the accumulation of peroxidized lipids that alter metabolic pathways and gene expression profiles in tissues and cells, it may indirectly regulate genes involved in tocopherol catabolism, lipid uptake, collagen synthesis, cellular adhesion, inflammation, the immune response, and cell signaling. Vitamin E affects several transcription factors, including peroxisome proliferator-activated receptor gamma (PPARγ), nuclear factor erythroid-derived 2 (NRF2), nuclear factor kappa B (NFκB), RAR-related orphan receptor alpha (RORα), estrogen receptor beta (ERβ), and the pregnane X receptor (PXR).

α-Tocopherol and its metabolites are believed to modulate the activity of several enzymes involved in signal transduction, including protein kinases and phosphatases, lipid kinases and phosphatases, and other enzymes involved in lipid metabolism, particularly those with inflammatory properties, such as lipoxygenases, cyclooxygenase-2, and phospholipase A₂. While the in vivo antioxidant properties of tocopherols have been questioned, this does not preclude a role in inhibiting oxidative enzymes, especially in relation to the immune system's function. For example, vitamin E regulates T cell function directly by affecting T cell membrane integrity, signal transduction, and cell division, and indirectly by influencing eicosanoids and related inflammatory mediators generated by other immune cells. Various tocopherols and tocotrienols have been shown to suppress COX-2 involvement in prostaglandin (PGD₂ and PGE₂) synthesis in lipopolysaccharide-activated macrophages.

In addition, the 13'-carboxy metabolite of α-tocopherol (α-T-13'-COOH) and other tocopherol ω-carboxylates are potent allosteric inhibitors of 5-lipoxygenase, a key enzyme in the biosynthesis of inflammatory leukotrienes. α-T-13'-COOH accumulates in immune cells and inflamed exudates, both in vitro and in vivo, in mice. It has even been suggested that the immune regulatory and anti-inflammatory functions of α-tocopherol depend on this endogenous metabolite. The structure of α-T-13'-COOH is shown in Figure \(\PageIndex{11}\).

α-Tocopherol stimulates the dephosphorylation enzyme, protein phosphatase 2A, which removes phosphate groups from protein kinase C, thereby deactivating it. The mechanism may involve vitamin E directly binding enzymes and competing with their substrates, or altering their activity through redox regulation. It may also compete for common binding sites within lipid transport proteins, thereby indirectly altering the traffic of lipid mediators and affecting their signaling functions and enzymatic metabolism. For example, it binds to albumin and a specific α-tocopherol-associated protein (TAP); in this latter form, it inhibits phosphoinositide 3-kinase. Vitamin E may also play a secondary role in stabilizing membrane structure by interacting with membrane enzymes to interfere with binding to specific membrane lipids, or by affecting membrane microdomains such as lipid rafts.

Evidence suggests that the biological activities of β-, γ-, and δ-tocopherols do not accurately reflect their antioxidant behavior; however, anti-inflammatory, antineoplastic, and natriuretic actions have been reported. Some non-antioxidant effects of γ-tocopherol in tissues, in relation to reactive nitrogen oxide species, have been observed; however, the specificity of these effects in vivo remains uncertain. In addition, researchers have attributed anti-inflammatory properties to a chain-shortened metabolite. Beneficial effects against cancer cells in vitro have been observed and attributed to scavenging reactive nitrogen species, as these effects are not observed with α-tocopherol. In contrast, vitamin E and its derivatives are believed to regulate tumor cells by activating the mitogen-activated protein kinase (MAPK) signaling pathway.

Tocotrienols have been shown to have neuroprotective effects and to inhibit cholesterol synthesis. They reduce breast cancer cell growth in vitro, possibly by influencing gene expression through interaction with the estrogen receptor-β. When administered with standard antitumor agents, such as chemotherapy drugs, or with natural compounds that exhibit anticancer activity, they are reported to exert a synergistic antitumor effect on cancer cells. γ-Tocotrienol is reported to induce apoptosis via endoplasmic reticulum stress, while α-tocotrienol may be neuroprotective by inhibiting lipoxygenase activity. Although mice have shown anti-obesity and anti-diabetic effects, clinical trials in humans have been inconclusive. These properties are largely distinct from those of tocopherols, and researchers are currently studying the pharmaceutical potential of tocotrienols against cancer, bone resorption, diabetes, and various diseases affecting the skin, cardiovascular system, and nervous system.

The biological functions of α-tocopheryl phosphate are slowly being revealed. In addition to serving as a possible storage or transport (water-soluble) form of tocopherol, it is involved in cellular signaling. It regulates many genes, including those involved in angiogenesis and vasculogenesis, in a manner distinct from α-tocopherol per se. Because it lacks a free hydroxyl group, it cannot act directly as an antioxidant, and some consider it the vitamin's biologically active form. It is certainly more active than α-tocopherol in several biological systems in vitro, so these effects cannot be attributed to the hydrolyzed molecule. In some instances, it is even antagonistic to α-tocopherol, for example, in its activity towards phosphatidylinositol 3-kinase. Activation requires a kinase, while a phosphatase is needed to make the system reversible, but neither has been identified. Synthetic phosphate derivatives of γ-tocopherol and α-tocopheryl succinate have potent anticancer properties.

Occurrence and Basic Metabolism of Carotenoids and Retinoids

The term ‘vitamin A’ is used to denote retinol (or all-trans-retinol, sometimes termed 'vitamin A₁'), together with a family of biologically active C₂₀ retinoids derived from this ('vitamers'). The structure of retinol is shown in Figure \(\PageIndex{12}\).

These are only found in animal tissues, where they are essential to innumerable biochemical processes. However, animals cannot synthesize them de novo, and their biosynthetic precursors are plant carotenoids with a β-ionone ring (provitamin A), which are C40 tetraterpenes; β-carotene is the most efficient. It is an orange-red pigment found in plant photosynthetic tissues and in seed oils. In the human diet in the developed world, plant sources tend to be less important than those from dairy products, meat, fish oils, and margarines, which provide vitamin A per se. However, carrots and spinach are good sources of the provitamin. In the U.K., for example, all vegetable spreads must be supplemented with the same level of vitamin A (synthetic retinol or β-carotene) as is found in butter. While most research has focused on retinoids, interest is growing in the biological activities of intact carotenoids in animal tissues.

Carotenoid biosynthesis in plants via isopentenyl diphosphate and dimethylallyl diphosphate has much in common with plant sterol biosynthesis, but this is too specialized a topic to cover in depth here. They have many important functions in plants, for example, during photosynthesis and as precursors to plant hormones, as discussed below. Some crop plants with increased carotene levels are being developed to prevent vitamin A deficiency in populations of developing countries, and further efforts are underway. Non-photosynthetic bacteria produce a different range of carotenoids, some with chain lengths other than C₄₀ (C₃₀-C₅₀).

In animals, including humans, dietary carotenoids such as β-carotene are solubilized in mixed micelles with other dietary lipids, aided by bile acids. They are absorbed in the intestines in their intact form, facilitated by specific receptor proteins. Dietary retinol and retinol esters are absorbed similarly in the intestines, but pancreatic lipase first hydrolyzes the latter. Conversion to retinoids, leading ultimately to retinol esters, occurs in the enterocytes, where dietary β-carotene is subjected to oxidative cleavage at its center, the first step of which is catalyzed by a cytosolic enzyme β-carotene-15,15'-oxygenase‑1 (BCO1), which is specific for carotenes with a β-ionone ring, to yield two molecules of all-trans-retinal, which is reversibly reduced by a retinol reductase to retinol. Xanthophyll carotenoids are mainly absorbed without cleavage. These reactions are shown in Figure \(\PageIndex{13}\).

Based on the incorporation of ¹⁸O into the products. The enzyme that introduces the oxygen atom, β-carotene-15,15'-oxygenase‑1 (BCO1), appears to be a dioxygenase, as both oxygen atoms from dioxygen are incorporated into the products. This contrasts with a possible mechanism in which only one oxygen atom from dioxygen is added, with the other coming from H₂O if the enzyme acted as a monooxygenase. Figure \(\PageIndex{14}\) shows how oxygen could be introduced in the reaction catalyzed by BCO1 through both possible mechanisms.

The dioxygenase mechanism on the right best accounts for the incorporation of ¹⁸O isotope of oxygen in dioxygen.

Figure \(\PageIndex{15}\) shows an interactive iCn3D model of the apocarotenoid cleavage oxygenase from Synechocystis, a Retinal-Forming Carotenoid Oxygenase (2BIW)

Figure \(\PageIndex{15}\): Retinal-Forming Carotenoid Oxygenase from Synechocystis (2BIW). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov...dttF958wFPAUP7

The enzyme has a bound carotene analog, (3R)-3-hydroxy-8'-apocarotenol. It contains an active site Fe²⁺ ion at the end of a hydrophobic tunnel. Four histidine side chains ligate the ion. On binding, three of the trans C=C bonds convert to a distorted cis-trans-cis conformation. The middle trans bond is proximal to the Fe^2+, which is ligated by dioxygen, the source of the incorporated oxygen atoms on cleavage.

Enterocytes incorporate any unchanged β-carotene and newly formed retinol esters into chylomicrons and release them into the lymphatic system and then into the bloodstream, where peripheral tissues take up some before most is absorbed by the liver. Some intact carotene and other carotenoids are transferred to lipoproteins (LDL and HDL) for transport in plasma, with assistance from specific binding and transport proteins. For example, carotene can be absorbed at the placental barrier and transferred to the fetus for conversion to retinoids, which are essential for development. Within hepatocytes, late endosomes hydrolyze retinol esters, releasing free retinol into the cytosol, where it can be returned to circulation, converted to retinoids, or transferred to hepatic stellate cells for storage in lipid droplets, the main body reservoir of vitamin A. In these specialized cells, retinol is esterified to form retinyl palmitate by transferring fatty acids from position sn-1 of phosphatidylcholine, mainly via a membrane-bound lecithin:retinol acyltransferase (LRAT) in the endoplasmic reticulum. Minor acyl-CoA-dependent pathways also exist, including acyl-CoA:retinol acyltransferase and diacylglycerol acyltransferase 1 (DGAT1); esterification is facilitated by binding to cellular retinol-binding protein type II (CRBP2).

Both retinol and retinoic acid are precursors of many metabolites, known as retinoids, required for specific tissue functions through enzymatic modification of functional groups and geometrical isomerization of polyene chains. In the liver, retinol activation first involves ester mobilization, followed by hydrolysis by retinol ester hydrolases, including carboxylesterase ES-10. Then, the reversible oxidation of retinol to retinal is carried out by one of several enzymes, including dehydrogenases and various cytochrome P450s, before some retinal is irreversibly oxidized to retinoic acid by enzymes with retinal dehydrogenase activity. Other tissues convert retinol to retinoic acid on demand through the same mechanisms. However, for vision, retinol esters serve directly as substrates for forming the visual chromophore 11-cis-retinal (see below). Retinyl-β-D-glucoside, retinyl-β-D-glucuronide, and retinoyl-β-D-glucuronide are naturally occurring, biologically active vitamin A metabolites found in fish and mammals. Indeed, the latter has activity similar to all-trans-retinoic acid without any unwanted side effects in some circumstances.

Cleavage of β-carotene at double bonds other than that in the center or of a wider range of other carotenoids occurs by the action of a related enzyme, β‑carotene-9',10'-dioxygenase (β‑carotene-oxygenase‑2 or BCO2), in mitochondria, which leads to the formation of similar molecules, i.e., β-apocarotenals and β‑apocarotenones of variable chain length. While these may exert distinctive biological activities in their own right, evidence suggests they can also be metabolized to form retinal.

In the aqueous environment within cells and in plasma, retinol, retinal, and retinoic acid are bound to retinoid-binding proteins (RBP), which solubilize, protect, and, in effect, detoxify them. These proteins also facilitate retinoid transport and metabolism; some are present only in specific tissues, and many are specific to particular retinoids and metabolic pathways. To prevent infiltration through the kidneys, retinol and holo-RBP associate in blood with a protein called transthyretin (TTR), which also carries thyroid hormones and is essential for their secretion. Normally, vitamin A circulates in plasma as a retinol:RBP:TTR complex with a 1:1:1 molar ratio. Unesterified retinol is the main form of the vitamin exported from the liver on demand, and it is transported in the blood in this bound form in VLDL, LDL, and HDL, with some coming directly from the diet in chylomicrons and their remnants. Peripheral tissues have specific receptors to take up what they require, likely after lipoprotein lipase hydrolyzes any esters to retinol. Then, retinol dissociates from the protein, forms a complex with a receptor (STRA6) at a target cell, and diffuses through the plasma membrane, a process driven by retinol esterification.

The RBP-TTR complex does not bind retinal or retinoic acid, although these bind to RBP on their own, and most low levels of retinoic acid transported in the blood are bound to albumin. Local retinoic acid levels result from an interplay among enzymes of synthesis, binding, and catabolism. For example, within cells, retinoic acid-binding proteins (CRABP1 and CRABP2) bind newly synthesized retinoic acid, increase its metabolism, and protect cells from excess.

In skin, 3,4-dehydroretinoids are synthesized from all-trans retinoids by the desaturase Cytochrome P450 27C1 with the assistance of cellular retinol-binding proteins (CRBPs). 3,4-Dehydroretinol, which is sometimes termed vitamin A₂, is shown in Figure \(\PageIndex{16}\).

Its derivative, 3,4‑dehydroretinal, is used as a visual chromophore in many cold-blooded vertebrates, including lampreys, fish, amphibians, and some reptiles (see below). Geranylgeranoic acid shares structural similarities with retinoic acid and is therefore termed an acyclic retinoid, although it lacks vitamin A activity. Animal tissues synthesize it from mevalonate, and it, together with its 2,3‑dihydro metabolite, has potent anticancer properties.

Retinol esters: A relatively small proportion of cellular retinoids is located in tissue membranes. Rather, retinol esters, mainly retinyl palmitate, are the main storage form of vitamin A and occur in many organs, including adipose tissue and testes, but chiefly in stellate cells of the liver and pancreas. The mechanism by which retinol is directed specifically to these cells and enters them before esterification is not known. Although hepatic stellate cells are much smaller and less abundant than hepatocytes (only 5 to 8% of all liver cells), they are characterized by cytoplasmic lipid droplets that contain 90-95% of the hepatic retinoids (and up to 80% of the body pool) in addition to other non-retinoid lipids; the lecithin:retinol acyltransferase is the only retinol ester synthase in this instance. Additionally, specialized cells in the eye store retinoids, which are essential for vision, as lipid droplets. When dietary retinol is limited, hepatic retinol ester stores are mobilized as retinol ester hydrolases are activated to maintain stable circulating retinol levels; hormone-sensitive lipase is the most important of these enzymes, although adipose tissue triacylglycerol lipase and lysosomal acid lipase are also involved.

Catabolism: All-trans-retinoic acid formation is irreversible, so its synthesis and degradation must be tightly regulated. In the first step of catabolism, various cytochrome P450 enzymes oxidize excess retinoic acid to more polar metabolites. Secondly, the water-soluble retinoic acid metabolites, including 4-hydroxy-, 4-oxo-, and 18-hydroxy-retinoic acids, are conjugated with glucuronic acid and can then be rapidly removed from circulation and eliminated from the body via the kidneys.

Retinoids and Vision

The structure of 11-cis-retinal, which we discussed in Chapter 11, is shown in Figure \(\PageIndex{17}\)

Retinoids are essential for vision, and we now understand how they work at the molecular level. In the eye, the transmembrane cell-surface STRA6 receptor of the retinal pigment epithelium mediates retinol uptake from the circulation; this pigmented monolayer of cells between the photoreceptors and choroid nourishes retinal visual cells, catalyzes the release of retinol from retinol-binding proteins, and transports it to the cytosol. The process by which light is converted to a signal recognized by the brain, sometimes termed the 'retinoid (visual) cycle', requires a two-cell system beginning in the retinal pigment epithelium and continuing in photoreceptor cells, i.e., retinal rod and cone cells in the eye that contain membranous vesicles that serve as light receptors. Roughly half of the proteins in these vesicles are the protein conjugate rhodopsin, which consists of the protein opsin and the retinoid 11-cis-retinal. Each step in the visual process requires specific binding or transport proteins, especially the interphotoreceptor retinoid-binding protein (IRBP).

All-trans-retinol is first converted to its ester by the enzyme lecithin:retinol acyltransferase in the RPE, and the products coalesce into lipid droplets, i.e., dynamic organelles termed 'retinosomes'. The next step involves a dual-purpose enzyme (RPE65) in the endoplasmic reticulum, which cleaves the O-alkyl bond (not a conventional hydrolysis reaction) in the retinol ester and, at the same time, changes the geometry of the double bond at position 11 of retinol from trans to cis. The 11-cis-retinol is then oxidized to 11-cis-retinal by 11-cis-retinal dehydrogenase (RDH5). The full cycle is shown in Figure \(\PageIndex{18}\ below.

The final part of the cycle occurs in the photoreceptor, where the 11-cis-retinal first reacts with opsin to produce the protein conjugate rhodopsin in a protonated form. When light activates rhodopsin, the cis double bond in the retinoid component isomerizes non-enzymatically to the 11-trans form, causing a conformational change that affects membrane permeability and influences calcium transport. This triggers further molecular changes that culminate in the release of opsin and all-trans-retinal, which initiates the nerve impulse and allows the brain to perceive light.

A second mechanism for 11-cis-retinal formation, which may function to ensure continuous visual responsiveness in bright light, involves the RPE-retinal G protein-coupled receptor (RGR), which can act as a retinaldehyde photoisomerase. Because RPE65 functions optimally in low light, RGR likely prevents photoreceptor saturation at high light levels, thereby facilitating vision in daylight. The isomerase RPE65 and the photoisomerase RGR work together to provide a sustained supply of the visual chromophore across different illumination levels.

The all-trans-retinal is removed from the photoreceptor either by reduction to all-trans-retinol by all-trans-retinol dehydrogenase 8 expressed in the outer segments of photoreceptors or after transport using a specific transporter (ABCA4), which provides phosphatidylethanolamine (PE) for conversion to the Schiff-base adduct, i.e., N-retinylidene-phosphatidylethanolamine, as shown in Figure \(\PageIndex{19}\).

It flips from the lumen to the cytosolic leaflet of the disc membrane. This process prevents non-specific aldehyde activity, thereby removing potentially toxic retinoid compounds from the photoreceptors. The adduct is a transient sink that dissociates, allowing cytoplasmic retinol dehydrogenase to reduce retinal back to all-trans-retinol. All-trans-retinol exits the photoreceptor and enters the retinal pigment epithelium, aided by binding to the retinoid-binding protein (IRBP), where it is converted back to a retinyl ester to complete the cycle and restore light sensitivity.

As a side reaction, non-enzymatic mechanisms may produce troublesome bis-retinoid adducts of PE (and further byproducts), which can accumulate with age and potentially affect vision. Lower organisms: Bacteriorhodopsin is the best-studied member of a family of opsins found in archaea, eubacteria, fungi, and algae. It is a protein with seven transmembrane helices that acts as an opto-electrical transducer, or light-gated active ion pump, capturing photon energy via its covalently bound chromophore, all-trans-retinal, converting it to 13-cis-retinal, and moving protons against their electrochemical gradient from the cytoplasm to the extracellular space. In Archaea, it is known as the "purple membrane" and can occupy a significant proportion of the organism's surface area.

Other Functions of Retinoids in Health and Disease

Beyond vision, retinoids are now recognized as essential for growth and development, reproduction, and resistance to infection. They are particularly important for epithelial cell function in the digestive tract, lungs, nervous system, immune system, skin, and bone at all life stages. They are required for the regeneration of damaged tissues, including the heart, and they appear to have potential as chemopreventive agents for cancer and for treating skin diseases such as acne. Under pathological conditions, stellate cells lose their retinoid content and transform into fibroblast-like cells, contributing to the fibrogenic response. A massive loss of retinoids accompanies cirrhosis of the liver, but it is not clear whether this is a cause or a symptom, and there appears to be confusion as to when supplementation may be helpful in this and other diseases of the liver. Like retinol and retinoic acid, the metabolite 9-cis-retinoic acid also has valuable pharmaceutical properties.

With so many conjugated double bonds, it is not surprising that carotenoids in general, and retinoids in particular, efficiently quench singlet oxygen and scavenge other reactive oxygen species. However, direct antioxidant properties are not believed to be important for general health in vivo, and it is unclear how relevant retinoid physical properties are to specific biochemical processes compared with their effects on signaling and gene transcription. One caveat is that retinoids may stimulate certain antioxidant genes, thereby exerting an indirect antioxidant function. Nutritional studies on carotenoid dietary supplements have sometimes suggested pro-oxidant activity. One explanation for the detrimental effects is that regeneration of the parent carotenoid or retinoid from the corresponding radical cation may be limited when reductant concentrations, such as those of ascorbic acid, are low.

Many retinol metabolites function as ligands that activate specific transcription factors for particular receptors in the cell nucleus. Thus, they regulate the expression of many genes (>500), including those essential for normal cell proliferation and differentiation, embryogenesis, a healthy immune system, and male and female reproduction. In the innate immune system, vitamin A is required for the differentiation of cells such as macrophages, neutrophils, and natural killer cells. All-trans-retinoic acid, by contrast, is involved in differentiating dendritic cell precursors. Retinoic acid and its 9-cis isomer are especially important in this context and are often considered the most important retinoids for functions other than vision. This structure of the cis-isomer is shown in Figure \(\PageIndex{20}\).

In essence, retinoic acid enters the nucleus with the aid of small intracellular lipid-binding proteins (CRABP2 and FABP5), which channel it to specific nuclear receptors, the retinoic acid receptors (RAR), of which there are three: RAR-α, β, and γ. These ligand-dependent transcription regulators function in vivo as heterodimers with retinoid X receptors (RXR) to process the retinoic acid signal by acting through polymorphic retinoic acid response elements (RAREs) in the promoter regions of responsive genes. Similarly, 9-cis-retinoic acid and 9-cis-13,14-dihydroretinoic acid are high-affinity RXR ligands in mice. Together with retinoic acid, these are also ligands for the farnesoid X receptor (FXR), which forms a heterodimer with RXR. This receptor complex is primarily involved in bile acid homeostasis, and, conversely, some evidence suggests that bile acids may regulate vitamin A homeostasis.

In addition, other nuclear receptors, such as the peroxisome proliferator-activated receptor PPARγ, form a heterodimer with the retinoid X receptor and are activated by retinoic acid to recruit cofactors. This complex then binds to the peroxisome proliferator-activated receptor response element (PPRE) on gene promoters, mainly regulating genes involved in lipid and glucose metabolism, including some involved in inflammation and cancer. To add to the complexity, retinoic acid has extranuclear, non-transcriptional effects, such as activating protein kinases and other signaling pathways.

It has also become evident that many of the functions of retinoids are mediated via the action of specific binding proteins (as discussed briefly above), which control their metabolism in vivo by reducing the effective or free retinoid concentrations, by protecting them from unwanted chemical attack, and by presenting them to enzyme systems in an appropriate conformation. With some tissues, retinol-bound RBP in the blood is recognized by the membrane protein STRA6, which transports retinol into cells, where it binds to an intracellular retinol acceptor, cellular retinol-binding protein 1 (CRBP1), and activates a signaling cascade that targets specific genes. In addition, a specific retinol-binding protein (RBP4) secreted by adipose tissue is involved in the development of insulin resistance and type 2 diabetes, possibly by affecting glucose utilization in muscle tissue, with clear implications for controlling obesity. In the eye, retinoic acid activity during development is controlled by binding to apolipoprotein A1.

All-trans-retinoic acid is effective against various types of human cancers, particularly in model systems and some clinical trials, because it specifically affects cell proliferation, differentiation, and apoptosis, and its relatively low toxicity at normal tissue levels is a benefit. For example, it induces complete remission in most cases of acute promyelocytic leukemia when administered in combination with other chemotherapy techniques. Similarly, 13-cis-retinoic acid has been used successfully in the treatment of children with high-risk neuroblastoma to reduce the risk of recurrence and increase long-term survival rates. However, the efficacy of similar treatments against other types of acute myeloid leukemia and solid tumors appears to be poor. Current efforts to better understand the mechanisms underlying anti-cancer activity are expected to lead to improved treatments. Synthetic analogs of retinoic acid, termed retinoids, which activate retinoic X receptors, also hold promise as anti-cancer agents.

Vitamin A deficiency in children and adult patients is usually accompanied by impairment of the immune system, leading to a greater susceptibility to infection and an increased mortality rate, often with growth retardation and congenital malformations. However, vitamin A deficiency in malnourished children is the major reason for childhood mortality in the underdeveloped world, causing over 650,000 early childhood deaths annually and pediatric blindness. This is doubly tragic, as it is so easily preventable. In adults, vitamin A deprivation affects the reproductive system, inhibiting spermatogenesis in males and ovulation in females. Unfortunately, distinguishing between the effects of vitamin A deficiency and primary defects of retinoid signaling is not always easy.

Functions of Xanthophylls and Other Carotenoids in Humans

Xanthophylls are plant C₄₀ tetraterpenes that differ from the carotenoids in having oxygen atoms in the ring structures (hydroxyl, oxo, or epoxyl). Lutein, zeaxanthin, and meso-zeaxanthin from dietary sources, such as green leafy vegetables and yellow and orange fruits and vegetables, are found specifically in the macula of the eye in humans and other primates, i.e., the functional center of the retina in a small central pit known as the macula lutea, where they enhance visual acuity and protect the eye from high-intensity, short-wavelength visible light. They are powerful antioxidants in a region vulnerable to light-induced oxidative stress. Binding proteins specific for lutein and zeaxanthin mediate the highly selective uptake of these carotenoids into the retina, but meso-zeaxanthin is mainly a metabolite of dietary lutein. Macular xanthophylls decrease the risk of age-related macular degeneration. In the brain, they may stimulate and maintain cognitive function in the elderly and assist with brain development in infants. Hydroxylated xanthophylls such as lutein, shown in Figure \(\PageIndex{21}\), occur both in the free form and esterified to fatty acids; the latter are hydrolyzed in the intestines when consumed by animals.

Animals absorb many other carotenoids from the diet. After being transported in lipoproteins, they undergo oxidative cleavage or other catabolic processes, partly in the intestines and partly in other tissues. Some carotenoids remain intact and are believed to act as antioxidants, while others may have specific anti-inflammatory properties. For example, carotenoids accumulate in the skin of mammals, where they may play antioxidant and photoprotective roles and affect moisture content, texture, and elasticity. Lycopene may protect against atherogenesis, coronary heart disease, and prostate cancer.

Functions of Carotenoids in Plants

Because carotenoids have a polyene chain of 9 to 11 conjugated double bonds, they can absorb light in the gap in chlorophyll absorption and function as additional light-harvesting pigments in plants. Their distinctive electronic-level arrangement enables them to transfer excitation energy from the carotenoid excited state to chlorophyll in the light-harvesting complex (photosystem II). Energy can also be transferred back from chlorophyll to carotenoids as a photoprotection mechanism. During photosynthesis, both light and oxygen produce damaging species, with reactive oxygen species (ROS) of special concern. Energy is transferred from chlorophyll to the carotenoid's polyene tail, where electrons move between the carotenoid bonds until the most balanced, or lowest-energy, state is reached. While carotenoids therefore have appreciable potential to act as antioxidants in plants, it is unclear how important this is from a practical functional standpoint. The length of the polyene tail of carotenoids determines which wavelengths of light the plant absorbs; those not absorbed are reflected, thereby determining the coloration.

Carotenoids are precursors for two plant hormones and a diverse set of apocarotenoids. For example, abscisic acid, shown in Figure \(\PageIndex{21}\), is a C₁₅ isoprenoid plant hormone, which is synthesized in plastids from the C₄₀ carotenoid zeaxanthin.

A series of enzyme-catalyzed epoxidations and isomerizations occurs, followed by cleavage of the intermediate product via dioxygenation and further oxidations to yield abscisic acid. Functioning via signaling cascades, abscisic acid regulates numerous biological processes in plants, especially developmental processes such as plant growth, seed and bud dormancy, embryo maturation and germination, cell division and elongation, floral growth, and stomatal closure. Plants must respond to environmental stresses, including drought, cold and heat stress, salinity, and tolerance to heavy metal ions. Similarly, strigolactones are C₁₅ oxidation products of carotenoids that regulate symbiosis between plants and arbuscular mycorrhizal fungi, as well as interactions with plant parasites.

Phytol

Phytol, or (2E,7R,11R)-3,7,11,15-tetramethyl-2-hexadecen-1-ol, i.e., a 20-carbon molecule with a 16-carbon chain and one double bond, is an acyclic diterpene alcohol synthesized in large amounts in plants as an essential component of chlorophyll, the most important photosynthetic pigment in plants and algae. Geranylgeranyl-diphosphate, synthesized in chloroplasts via the 4-methylerythritol-5-phosphate (non-mevalonate) pathway, is the primary precursor of phytol after geranylgeranyl reductase reduces three double bonds, and this can occur before or after attachment to chlorophyll, depending on species. Chlorophyll dephytylase (CLD1) is the enzyme in plants responsible for chlorophyll hydrolysis and phytol release. The reactions are shown in Figure \(\PageIndex{22}\).

Little free phytol is present in plant tissues. However, some phytol esters of fatty acids may form, especially during nitrogen deprivation or senescence, when chlorophyll is degraded, fatty acids are released from glycerolipids, and a phytol ester synthase is induced as part of a detoxification and recycling process. Bell peppers and rocket salad are especially rich sources under normal conditions. In addition, phytol, as its diphosphate, is utilized for the synthesis of tocopherols (vitamin E) and phylloquinol (vitamin K - see below), and the precursor geranylgeraniol and its fatty acid ester occur in small amounts in some plant species. It is the biosynthetic precursor of tocotrienols and the highly unsaturated carotenoids (and hence of retinoids). Phytenal has been isolated as an intermediate in phytol catabolism in plants, but subsequent steps remain uncertain, although stressed plants have been shown to contain phytanoyl-CoA. Because phytenal is highly reactive and potentially toxic through protein interactions, competing pathways must keep its accumulation low.

In ruminants, rumen microorganisms hydrolyze chlorophyll, releasing free phytol. This does not occur in humans, but some phytol may be ingested with plant foods, either in its free form or as phytol esters, and can be absorbed from the intestines. Within animal tissues, phytol is oxidized to phytanic acid. Phytol and/or its metabolites have been reported to activate the transcription factors PPARα and the retinoid X receptor. In mice, oral phytol induces substantial peroxisome proliferation in many organs.

Plastoquinone

A molecule that is related to the tocopherols, plastoquinone, is found in cyanobacteria and plant chloroplasts, and it is produced in plants by analogous biosynthetic pathways to those of tocopherols in the inner chloroplast envelope with solanesol diphosphate as the biosynthetic precursor of the side chain; there appears to be a somewhat different mechanism in cyanobacteria. The molecule is sometimes designated as 'plastoquinone-n' (or PQ-n), where 'n' is the number of isoprene units, which can vary from 6 to 9. Its structure is shown in Figure \(\PageIndex{23}\).

Plastoquinone plays a crucial role in photosynthesis by linking photosystems I and II in electron transport, thereby generating an electrochemical proton gradient across the thylakoid membrane. This provides energy for adenosine triphosphate (ATP) synthesis. Reduced dihydroplastoquinone (plastoquinol) transfers electrons to photosynthesis enzymes before a specific cytochrome complex reoxidizes it. The redox state of the plastoquinone pool regulates the expression of many genes encoding photosystem proteins. X-ray crystallography studies of photosystem II from cyanobacteria reveal that two molecules of plastoquinone form two membrane-spanning branches. In addition, plastoquinone has antioxidant activity comparable to tocopherols, protecting especially against excess light energy and photooxidative damage. Similarly, in thylakoid membranes, plastoquinol can scavenge superoxide, producing H₂O₂. Plastoquinone is a cofactor that participates in the desaturation of phytoene in carotenoid biosynthesis and is a biosynthetic precursor of plastochromanols. With its many functions, plastoquinone links photosynthesis in plants to metabolism, light acclimation, and stress tolerance.

Plastoquinone-9, together with phylloquinone, tocopherol, and plastochromanol-8, is stored in plastoglobuli, lipoprotein-like micro-compartments, which enable exchange with the thylakoid membrane and are also involved in chlorophyll catabolism and recycling. It has been suggested that the redox state of the plastoquinone pool is the main redox sensor in chloroplasts, initiating many physiological responses to environmental changes, particularly those related to light intensity, by regulating the expression of chloroplast genes.

Ubiquinone (Coenzyme Q)

The ubiquinones, also known as coenzyme Q (CoQ) or mitoquinones, have clear biosynthetic and functional relationships with plastoquinone and are found in all domains of life (hence the name). They have a 2,3‑dimethoxy-5-methylbenzoquinone nucleus and a side chain of six to ten isoprenoid units; the human form illustrated below has ten such units (coenzyme Q₁₀), i.e., 2,3-dimethoxy-5-methyl-6-decaprenyl-1,4-benzoquinone, while the rat has nine, Escherichia coli has eight, and Saccharomyces cerevisiae has six. In plants, ubiquinones tend to have nine or ten isoprenoid units. Forms with a second chromanol ring, resembling the structures of tocopherols, are also produced (ubichromanols), but not in animal tissues. Yeast fermentation produces them on an industrial scale for pharmaceutical use. Because of their hydrophobic properties, ubiquinones are located entirely in membrane bilayers in most eukaryotic organelles, probably at the mid-plane.

Ubiquinones are synthesized de novo in mitochondria in most cells in animal, plant, and bacterial tissues by a complex sequence of reactions from the essential amino acid phenylalanine and then tyrosine to generate p-hydroxybenzoic acid, which is the key precursor that is condensed with the polyprenyl unit (from the cholesterol synthesis pathway) via a specific transferase; this is followed by decarboxylation, hydroxylation, and methylation steps, depending on the specific organism, although some of the required enzymes have yet to be fully characterized. In Escherichia coli, biosynthesis does not occur in a membrane environment, as previously thought. Instead, the seven proteins that catalyze the last six reactions of the biosynthetic pathway, following attachment of the isoprenoid tail, form a stable complex, or metabolon, in the cytoplasm, enabling modification of hydrophobic substrates in a hydrophilic environment.

In mitochondria, coenzyme Q is present both as the oxidized (ubiquinone) and reduced (ubiquinol) forms, as shown in Figure \(\PageIndex{24}\).

Figure \(\PageIndex{24}\): Ubiquinone-ubiquinol interconversion

Ubiquinones are essential components of the respiratory electron transport system, possibly as part of supramolecular complexes, taking part in the oxidation of succinate or NADH via the cytochrome system to generate the protonmotive force used by the mitochondrial ATPase to synthesize ATP. In this process, coenzyme Q transfers electrons from the various primary donors, including complex I, complex II, and the oxidation of fatty acids and branched-chain amino acids, to the oxidase system (complex III), while simultaneously transferring protons to the outside of the mitochondrial membrane, resulting in a proton gradient across the membrane. As a consequence, it is reduced to ubiquinol. Thus, it is an essential component of the cycle that generates the proton motive force driving ATP production via oxidative phosphorylation. In yeast, one coenzyme Q binding protein (COQ10), and in humans, two related proteins (COQ10A and COQ10B) may serve as chaperones or transporters during this process. Mitochondrial coenzyme Q is also implicated in reactive oxygen species production through a mechanism involving the formation of superoxide from ubisemiquinone radicals, thereby contributing to some of the oxidative damage underlying many degenerative diseases. In this role, it acts as a pro-oxidant. It is most abundant in organs with a high metabolic rate, such as the heart, kidneys, and liver.

In its reduced form (ubiquinol), it acts as an endogenous antioxidant in non-mitochondrial cellular membranes and plasma lipoproteins, making it the only lipid-soluble antioxidant synthesized endogenously. It inhibits lipid peroxidation in biological membranes and serum low-density lipoproteins, and it may also protect membrane proteins and DNA against oxidative damage. Ferroptosis suppressor protein 1 (FSP1) replenishes ubiquinol, which protects by combating lipid peroxidation that drives ferroptosis. The mechanism involves FSP1 recruitment to the plasma membrane after myristoylation, where it functions as an oxidoreductase that reduces ubiquinone to ubiquinol, a lipophilic radical-trapping antioxidant that halts lipid peroxide propagation. In this manner, it regulates cellular redox status and cytosolic oxidative stress, thereby serving as a key regulator of apoptosis. Other NAD(P)H dehydrogenases with CoQ reductase activity include cytochrome b5 reductase and NQO1 (NAD(P)H:quinone oxidoreductase).

Although ubiquinone has only about one-tenth of the antioxidant activity of vitamin E (α-tocopherol), it can enhance its effects by regenerating it from its oxidized form back to its active, fully reduced state (similarly with vitamin C). However, ubiquinones and tocopherols appear to exhibit both cooperative and competitive effects under different conditions. Similarly, in bacteria and other prokaryotes, ubiquinones participate in many redox reactions, notably in the respiratory electron transport system but also in other enzyme reactions that require electron donation, including disulfide bond formation.

Coenzyme Q has many other functions unrelated to its antioxidant activity. Enzymes in mitochondria use coenzyme Q to link the respiratory chain to other metabolic pathways, including fatty acid β-oxidation as an electron acceptor, de novo nucleotide biosynthesis, amino acid oxidation (glycine, proline, glyoxylate, and arginine), and sulfide detoxification. Through these activities, coenzyme Q may indirectly modulate metabolic pathways located outside the mitochondria. Some evidence also suggests that coenzyme Q may help regulate redox-dependent cell signaling and gene expression, particularly by repressing inflammatory gene expression. Regarding its anti-inflammatory properties, clinical studies suggest that coenzyme Q10 supplementation significantly reduces levels of inflammatory mediators, including C-reactive protein, interleukin-6, and tumor necrosis factor-alpha (TNF-α). Additionally, it regulates mitochondrial permeability and, in relation to pyrimidine nucleotide biosynthesis, is essential for DNA replication and repair.

Dietary ubiquinone, i.e., that in food or dietary supplements, is absorbed by enterocytes via a process of “passive facilitated diffusion”, probably requiring a carrier molecule, before incorporation into chylomicrons for transport to the liver. This eventually leads to elevated ubiquinol levels in the blood, especially in LDL and VLDL lipoproteins, presumably because the lymphatic system has reduced levels of the oxidized form. Consequently, enhanced protection against lipid peroxidation has been reported, with health benefits, particularly related to cardiac function, sperm motility, and neurodegenerative diseases. For example, CoQ levels in both plasma and the heart correlate with heart failure in patients, and clinical trials of dietary supplementation have shown promising results. This may be particularly important in the elderly or in patients taking statins, as endogenous synthesis declines. CoQ₁₀ deficiency syndrome is associated with inherited pathological diseases and is defined by decreased CoQ₁₀ content in muscle and/or cultured skin fibroblasts. Early clinical trials with CoQ10 and a synthetic analog, idebenone, against various neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and others, are encouraging.

Phylloquinone and Menaquinones (Vitamin K)

Phylloquinone, or 2-methyl-3-phytanyl-1,4-naphthoquinone, is synthesized in the inner chloroplast envelope of cyanobacteria, algae, and higher plants by a mechanism analogous to that of the tocopherols, i.e., from chorismate in the shikimate pathway with a prenyl side chain derived from phytyldiphosphate. In this membrane, it is a key component of the photosystem I complex, where it receives an electron from the chlorophyll a acceptor molecule and then donates it to the membrane-associated iron-sulfur protein acceptor cluster. In a clear parallel to the plastoquinones (above), two molecules of phylloquinone form two membrane-spanning branches, as shown by X-ray crystallography studies of photosystem I from cyanobacteria. Plastoglobules associated with the thylakoid membrane are believed to function as a reservoir for excess phylloquinone and may also function in its metabolism. Edward A. Doisy and Henrik Dam received the Nobel Prize in Physiology or Medicine in 1943 for discovering vitamin K and determining its chemical structure. The structure of vitamin K is shown in Figure \(\PageIndex{25}\).

Menaquinones are related bacterial products that function in bacterial respiratory and photosynthetic electron transport chains. They have a variable number (4 to 10) of isoprenoid units in the tail, and they are sometimes designated ‘MK-4’ to ‘MK-10’. Unlike phylloquinone, these are usually highly unsaturated. In some species, methyl or other groups are attached to the naphthoquinone moiety. Remarkably high concentrations of menaquinones are present in the membranes of some extremophiles, such as haloarchaea, where they are suggested to act as ion-permeability barriers and as a powerful shield against oxidative stress, in addition to their roles as electron and proton transporters.

Phylloquinone is an essential dietary component for animals and is commonly referred to as 'vitamin K₁'. Animals must obtain it from green plant tissues, where it occurs at 400-700 μg/100 g, or from seed oils. Menaquinones, whose main dietary sources are cheese and yogurt, also have vitamin K activity and are called 'vitamin K₂'. They account for about 10-25% of the vitamin K content of the Western diet. A synthetic saturated form of this, which is used in animal feeds, is known as 'vitamin K₃ or menadione', though strictly speaking it is not a vitamin but a provitamin in that it can be converted to the menaquinone MK-4 in animal tissues by addition of a phytyl unit; it is too toxic for human nutrition. Vitamin K forms are absorbed from the intestines and transported in plasma as lipoproteins, similar to the other fat-soluble vitamins. Different tissues have different storage capacities and, presumably, different requirements for the various forms of vitamin K. Because a large proportion is excreted, there appears to be a requirement for constant intake. The liver takes up vitamin K₁ rapidly, but vitamin K₂ remains in the plasma much longer and may be the main source of the vitamin in peripheral tissues. In addition, some dietary phylloquinone is converted to menaquinone-4 in animals, although its quantitative significance remains unclear; the mechanism involves conversion to menadione in the intestines, followed by transport to tissues, where a specific prenyltransferase attaches a geranylgeranyl side chain.

The primary role of vitamin K in animal tissues is to act as a cofactor for the vitamin K-dependent enzyme γ-glutamyl carboxylase in the liver's endoplasmic reticulum. It catalyzes the post-translational carboxylation of glutamate residues to form γ-carboxyglutamic acid in proteins such as prothrombin. This activation of prothrombin and three related proteins promotes blood clotting. The γ-carboxyglutamic acid residues are located at the Ca²⁺ binding site and are vital for the enzyme's activity. Phylloquinone must first be converted to the reduced form, phylloquinol, the enzyme's actual cofactor; molecular oxygen and carbon dioxide are also required. Phylloquinol donates a hydrogen to the glutamic acid residue, which is then oxidized to 2,3-epoxyphylloquinone. A further enzyme, vitamin K epoxide reductase, regenerates phylloquinone by reduction of the epoxide in a dithiol-dependent reaction so that this can be re-utilized many times ("the vitamin K cycle", shown below in Figure \(\PageIndex{26}\)).

Menaquinones undergo the same reaction cycle. By interfering with the last step in the metabolic cycle, warfarin, the rodenticide, prevents blood clotting. Similarly, a deficiency in vitamin K results in the inhibition of blood clotting and can lead to brain hemorrhaging in malnourished newborn infants. However, this is not observed in adult humans, presumably because intestinal bacteria produce sufficient amounts to meet our needs. Vitamin K-dependent proteins also play important roles in the central and peripheral nervous systems, and vitamin K influences sphingolipid biosynthesis in the brain.

Unsaturated isoprene units, rather than phytol, are used to synthesize menaquinones, which differ from phylloquinone in chemical structure and pharmacokinetics. Vitamin K₂ (especially MK-4) now appears to have several distinct actions, some of which are tissue-specific. For example, osteocalcin is a γ-carboxyglutamic acid-containing protein that forms a strong complex with the mineral hydroxyapatite (calcium phosphate) in bone; it must be carboxylated to function properly, and vitamin K₂ appears to be particularly important in this process. Vitamin K₂ also regulates bone remodeling by osteoclasts to remove old or damaged bone and replace it with new bone. Vitamin K₂ also helps regulate vascular calcification, cell growth, and apoptosis. Side effects of using anticoagulants that interfere with vitamin K can include osteoporosis and an increased risk of vascular calcification. Although careful dosage control is necessary, vitamin K₂ may be a useful adjunct in treating osteoporosis and may reduce morbidity and mortality in cardiovascular disease by reducing vascular calcification.

The liver catabolizes excess vitamin K₁ and menaquinones via a common degradative pathway that shortens the isoprenoid side chain to yield carboxylic acid aglycones such as menadiol, which can be excreted in bile and urine as glucuronides or sulfates.

Dolichols and Polyprenols

Polyisoprenoid alcohols, such as dolichols, are ubiquitous, though minor, components of the membranes of most living organisms, from bacteria to mammals. They are hydrophobic linear polymers consisting of up to 20 isoprene residues (or 100 carbon atoms; or many more in plants, especially), linked head-to-tail, with a hydroxyl group at one end (α-residue) and a hydrogen atom at the other (ω-end). In indolichols (or dihydropolyprenols), the double bond in the α-residue is hydrogenated, distinguishing them from polyprenols with a double bond in the α-residue. Their structures are shown in Figure \(\PageIndex{27}\).

Polyisoprenoid alcohols are further differentiated by the geometrical configuration of the double bonds into three subgroups, i.e., di-trans-poly-cis, tri-trans-poly-cis, and all-trans. For many years, it was assumed that polyprenols were present only in bacteria and plants, particularly in photosynthetic tissues, whereas dolichols were found only in mammals and yeasts. However, dolichols can also occur at low levels in bacteria and plants, while polyprenols have been detected in animal cells.  Solanesol is a related, distinctive plant product with only trans double bonds and serves as a precursor to plastoquinone.

Within a given species, components of one chain length may predominate, but other homologs are usually present. The chain length of the main polyisoprenoid alcohols varies from 11 isoprene units in eubacteria to 16 or 17 in Drosophila, 15 and 16 in yeasts, 19 in hamsters, and 20 in pigs and humans. In plants, the range is from 8 to 22 units; however, some plant species have an additional class of polyprenols with up to 40 units. In tissues, polyisoprenoid alcohols can be present in the free form, esterified with acetate or fatty acids, phosphorylated, or monoglycosylated and phosphorylated (in various forms), depending on the species and tissue. Polyisoprenoid alcohols, per se, do not form bilayers in aqueous solution, but rather a type of lamellar structure. However, they are found in most membranes, especially the plasma membrane of liver cells and the chloroplasts of plants.

Dolicholic acids, i.e., related molecules with a terminal carboxyl group and containing 14–20 isoprene units, have been isolated from the substantia nigra of the human brain. However, they were barely detectable in the pig brain.

The biosynthesis of the basic building block of dolichols, such as isopentenyl diphosphate, occurs via either the mevalonate pathway or the more recently described methylerythritol phosphate pathway, as previously discussed in relation to cholesterol biosynthesis. Farnesyl pyrophosphate is the primary precursor in polyprenol biosynthesis and the branch point in sterol/isoprene biosynthesis, depending on the organism (see a further note below). Subsequent formation of the linear prenyl chain is accomplished by cis-prenyl transferases that catalyze the condensation of isopentenyl pyrophosphate and farnesyl pyrophosphate, followed by elongation of the allylic prenyl diphosphate chain. The end products are polyprenyl pyrophosphates, which are first dephosphorylated to polyprenol phosphate and then to the free alcohol. Finally, a specific reductase identified in human tissues catalyzes the reduction of the double bond at position 2 to produce dolichols. There is a family of cis-prenyl transferases, present in both eukaryotes and bacteria, that, in addition to the synthesis of dolichols, can catalyze the formation of isoprenoid carbon skeletons from neryl pyrophosphate (C₁₀) to natural rubber (C_>10,000), including the polyisoprenoid phosphates involved in protein glycosylation as discussed in the next section. The reactions in the synthesis of dolichol are shown in Figure \(\PageIndex{28}\).

Although polyprenols and dolichols were initially regarded as secondary metabolites, they are now recognized to have significant biological functions. Glycosylation of asparagine residues is the primary protein modification in all three domains of life, and phosphorylated polyisoprenoids, including dolichols, are crucial to this process (see next section). Some evidence also suggests that free dolichol may have a beneficial antioxidant function in cell membranes.

Polyisoprenoid Phosphates and Glycosylation of Proteins

Glycosylated phosphopolyisoprenoid alcohols carry oligosaccharide units for transfer to proteins. As glycosyl donors, they serve as substrates for glycosyl transferases in glycan biosynthesis, similar to cytosolic sugar nucleotides. They differ from the latter in location: the lipid portion is in the endoplasmic reticulum membrane, and the oligosaccharide portion is on either the cytosolic or lumenal face of the membrane. The product's degree of unsaturation and chain length are important for recognition by the enzymes in the next stage of the pathway.

Dolichol phosphates: In eukaryotes, N-glycosylation begins on the cytoplasmic side of the endoplasmic reticulum, where carbohydrate moieties are transferred from nucleotide-activated sugar donors, such as uridine diphosphate N-acetylglucosamine, onto dolichol phosphate. Then, N-acetylglucosamine phosphate is added to give dolichol-pyrophosphate linked to N-acetylglucosamine, to which a second N-acetylglucosamine unit is added, followed by five mannose units; the last is added by dolichol phosphate mannose synthase, which is also essential for GPI-anchor biosynthesis. The dolichol-PP glycan is shown in Figure \(\PageIndex{29}\).

The resulting dolichol-pyrophosphate heptasaccharide is then flipped across the endoplasmic reticulum membrane to the luminal face with the aid of a “flippase”. Glycosyltransferases add four additional mannose and three glucose residues to the oligosaccharide chain, using dolichol-phospho-mannose and dolichol-phospho-glucose as donors.  These donors are also synthesized on the cytosolic face of the membrane and flipped across to the luminal face. In humans, the final lipid product is a C₉₅-dolichol pyrophosphate-linked tetradecasaccharide, whose oligosaccharide unit is transferred from the dolichol carrier onto specific asparagine residues on a developing polypeptide in the membrane. The carrier dolichol-pyrophosphate is dephosphorylated to dolichol-phosphate, then diffuses or is flipped back across the endoplasmic reticulum to the cytoplasmic face.

The Archaea utilize dolichol in the synthesis of lipid-linked oligosaccharide donors, with both dolichol phosphate (Euryarchaeota) and pyrophosphate (Crenarchaeota) serving as carriers. These donors can have variable numbers of isoprene units, many of which can be saturated. In the haloarchaeon Haloferax volcanii, for example, a series of C₅₅ and C₆₀ dolichol phosphates with saturated isoprene subunits at the α- and ω-positions is involved in the glycosylation reaction of target proteins. Similarly, methanogens also use similar lipid carriers for oligosaccharide units. Archaea, of course, use isoprenyl ethers linked to glycerol as major membrane lipid components in addition to unusual carotenoids such as the C₅₀ bacterioruberins. In many of these species, isoprenoid biosynthesis occurs via the 'classical' mevalonate pathway; however, in other species, certain aspects of this pathway differ.

Undecaprenylphosphate: Most other bacteria use undecaprenyl-diphosphate-oligosaccharide as a glycosylation agent in a similar way for the biosynthesis of peptidoglycan, the main component of most bacterial cell walls and a structure unique to bacteria, of many other cell-wall polysaccharides, including lipopolysaccharides, O-antigenic polysaccharides, and capsular polysaccharides, and of N-linked protein glycosylation in both Gram-negative and Gram-positive bacteria. Undecaprenyl phosphate (a C₅₅ isoprenoid), also referred to as bactoprenol, is the essential lipid intermediate. Its structure is shown in Figure \(\PageIndex{30}\).

It differs from dolichol phosphates mainly because its terminal unit is unsaturated and is synthesized by adding eight isopentenyl pyrophosphate units to farnesyl pyrophosphate, a reaction catalyzed by undecaprenyl pyrophosphate synthase in the cytoplasm, followed by removal of a phosphate group. Undecaprenyl phosphate is required for glycan synthesis and transport for external polymer formation. Thus, membrane-embedded or membrane-associated enzymes covalently transfer glycans to the carrier lipid using nucleotide-activated precursors. For example, the carrier lipid with GlcNAc-MurNAc-peptide monomers (i.e., lipid II) is hydrophilic and is then transported across the cytoplasmic membrane to external sites for peptidoglycan formation. It is also required for the synthesis of lipoteichoic acids and lipopolysaccharide O-antigens.

Lipid II: Undecaprenyl diphosphate-MurNAc-pentapeptide-GlcNAc, often termed lipid II, is the last major lipid intermediate in peptidoglycan cell wall construction in bacteria (Lipid I is the biosynthetic precursor lacking the N-acetylglucosamine residue). Its structure is shown below in Figure \(\PageIndex{31}\).

Figure \(\PageIndex{31}\): Lipid II

This molecule must be translocated from the cytosol to the exterior membrane of the organism, and three different protein classes have been identified that can accomplish this; ATP-binding cassette (ABC) transporters are best characterized. Once across the membrane, lipid II is cleaved to provide the MurNAc-pentapeptide-GlcNAc monomer, which polymerizes and crosslinks to form the complex peptidoglycan polymer that provides strength and shape to bacteria. A membrane-integrated member of the type II phosphatidic acid phosphatase family hydrolyzes the remaining undecaprenyl-pyrophosphate to undecaprenyl phosphate, which an as-yet-unidentified transport mechanism recycles back to the interior of the membrane. The turnover rate is very high, so the lipid II cycle is considered to be the rate-limiting step in peptidoglycan biosynthesis. Evidence also suggests that lipid II helps organize cytoskeletal proteins in the inner leaflet of the cytoplasmic membrane. Because of its highly conserved structure and surface membrane accessibility, the proteins involved in lipid II synthesis and transport are important targets for developing novel antibiotics. Figure \(\PageIndex{xx}\):

In a few prokaryotes, the membrane intermediate has a polyprenyl-monophosphate-glycan structure instead of lipid II, and undecaprenyl-phosphate-L-4-amino-4-deoxyarabinose is involved in lipid A modification in Gram-negative bacteria, for example. There are obvious parallels with the involvement of glycosylated phosphopolyisoprenoid alcohols as carriers of oligosaccharide units for transfer to proteins and as glycosyl donors in higher organisms (see above).

β‑D‑Mannosyl phosphomycoketide is an isoprenoid phosphoglycolipid found in the cell walls of Mycobacterium tuberculosis, whose lipid component is a C32-mycoketide with a saturated oligoisoprenoid chain and five chiral methyl branches. It acts as a potent antigen that activates T cells upon presentation by CD1c. Its structure is shown in Figure \(\PageIndex{32}\).

Farnesyl Pyrophosphate and Related Compounds

Farnesyl pyrophosphate is a key intermediate in the biosynthesis of sterols such as cholesterol, and it is the donor of the farnesyl group in the biosynthesis of dolichols and polyprenols (see above) as well as for the isoprenylation of many proteins (see the web page on proteolipids). However, it also mediates various biological reactions through interaction with a specific receptor. It is synthesized through two successive phosphorylations of farnesol.

Presqualene diphosphate is unique among isoprenoid phosphates because it contains a cyclopropylcarbinyl ring. In addition to being a biosynthetic precursor of squalene and, thence, of cholesterol, it is a natural anti-inflammatory agent that functions by inhibiting phospholipase D activity and the generation of superoxide anions in neutrophils.

Their structures are shown in Figure \(\PageIndex{32}\).