Carotenoids are one of the largest classes of natural pigments synthesized in all photosynthetic organisms (plants, algae and cyanobacterial) and in some non- photosynthetic organisms such as bacteria and fungi (Burkhardt et al., 1997). Mammals including humans cannot synthesize carotenoids even though they are essential source of retinoids and vitamin A (Botella-Pavia et al., 2004) and are responsible for the colour of familiar animals such as lobster, flamingo and fish (Klaui and Bauernfeind, 1981). In plants the carotenoid pigments are synthesized in the plastids. They accumulate primarily in the chloroplasts of the photosynthetic membranes and senescing leaves or in the chromoplasts of ripening fruits, flower petals or other tissues such as carrot root (Cunningham and Gantt, 1998; Bartley and Scolnik, 1995). In some cases, carotenoids also can be formed in the amyloplasts of plant storage tissues such as maize and potato (Burkhardt et al., 1997). Most of the carotenoids important in photosynthetic organisms are
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xanthophylls or oxygenated carotenoids (Goodwin, 1980). The sesqui- and triterpenoids are produced in the cytoplasm whereas mono-, di- and tetraterpenoids are produced in the plastids (Kleinig, 1989). The dihydroxy carotenoid zeaxanthin is thought to play a central role in the nonradiative dissipation of light energy. Zeaxanthin is formed from β-carotene by hydroxylation serves as the substrate for biosynthesis of many other important xanthophylls (Demmig-Adams et al., 1996). Lutein, violaxanthin and neoxanthin are the essential components of the light-harvesting antennae where they absorb photons and transfer the energy to chlorophyll as well as assisting in the harvesting of light in the range of 450-570 nm (Van den Berg et al., 2000).
At present, more than 600 different carotenoid structures have been identified with β-carotene is the most prominent number in this group (Pfander, 1987). The typical carotenoids found in plant chloroplasts are lutein, zeaxanthin, antheroxanthin, violaxanthin and neoxanthin and in chromoplasts are capsanthin, capsorubin, bixin, crocetin and citraurin (Van den Berg et al., 2000). In this context, the natural biological functions and actions of carotenoids are based on the physical and chemical properties of the molecules to ensure its fits into cellular and subcellular structures in the correct location and orientation to allow its function efficiently and to determine the photochemical properties and chemical reactivity that form the basis of these functions (Britton, 1995).
12 1.6 Carotenoid biosynthesis
Carotenoids are biosynthesized by the well known isoprenoids pathway of mevalonic acid (Figure 1.1) and often commences with the formation of phytoene (Figure 1.2) from condensation of two GGPP molecules (Taylor and Ramsay, 2005).
Figure 1.1: Early stage and formation of isopentenyl diphosphate by the MVA-independent pathway. Abbreviations: GA3P, glyceraldehyde-3-phosphate; TPP, thiamine pyrophosphate; DXP, 1-deoxy-D-xylulose 5-phosphate; MEP, 2-C-methyl-D-erythritol 4-phosphate; CDP-ME, 4-(cytidine 5´-diphospho)-2-C-methyl-D- erythritol; CDP-MEP, 2-phospho-4-(cytidine 5´-diphospho)-2-C- methyl-D-erythritol; MEP-cPP, 2-C-methyl-D-erythritol-2,4- cyclodiphosphate; HMBPP, 1-hydroxy-2-methyl-2-(E)-butenyl 4- phosphate; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; DXPS, 1-deoxy-D-xylulose 5-phosphate synthase; DXR, 1-deoxy-D-xylulose 5-phosphate reductoisomerase; MCT, 2-C-methyl-D-erythritol 4-phosphate cytidyl transferase; CMK, 4- (cytidine 5´-diphospho)-2-C-methyl-D-erythritol kinase; MCS, 2- C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; HDS, 1- hydroxy-2-methyl-2-(E)-butenyl 4-phosphate synthase and IPPi, isopentenyl diphosphate isomerase.
Figure 1.2: Condensation or phytoene synthesis from IPP and DMAPP. Abbreviations: IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate; PPPP, prephytoene diphosphate; GPS, geranyl diphosphate synthase; GGPS, geranylgeranyl diphosphate synthase and PSY, phytoene synthase. OH O O + O OH OP OP O OH OH OP OH OH OH OH OH OH O CDP O P OH OH O CDP O P OH OH O P O OH OPP OPP OPP PYRUVATE DXPS TPP DXR NADPH Mn2+ MCT DXP MEP CDP-ME MCK CDP-MEP MCS MEP-cPP HDS HMBPP IDS IDS DMAPP IPP IPPi Mg2+ Mg2+ GA 3P ATP CTP CH3 H CH2OPP CH2OPP CH2OPP CH2OPP CH2OPP CH2OPP DMAPP IPI IPP IPP IPP GPP IPP FPP GGPP GGPP PPPP PHYTOENE C5 C10 C15 C20 C40 C40 GPS FPS GGPS PSY
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Four desaturation forms (Figure 1.3), sequentially, phytofluene, ζ-carotene, neurosporene and finally red coloured lycopene are converted and derived from colourless phytoene. In the final stage of this reaction, one of two alternative hydrogen atoms is lost, stereospecifically, and this determines whether the product is trans (all-E) or 15-cis (15Z) phytoene (Britton, 1989). The cyclization of lycopene (Figure 1.4) with lycopene cyclases, β-(LCYB) and ε-(LCYE), is a significant branch-point in carotenoid biosynthesis. These rings are formed by separate pathways. On one branch a single enzyme LCYB can catalyses the introduction of two β rings into lycopene to form β-carotene and in the other branch of the pathway LCYE can only incorporate one ε-ring forming δ-carotene. In order to form α-carotene both LCYE and LCYB must act.
Figure 1.3: Desaturation and isomerization of phytoene. Abbreviations: PDS, phytoene desaturase; ZDS, ζ-carotene desaturase and CRTISO, carotene isomerase.
Figure 1.4: Cyclisation of lycopene. Abbreviations: LCYE, ε- cyclase; LCYB, β-cyclase.
PHYTOENE PDS -2H DI-CIS-PHYTOFLUENE PDS -2H CIS-Z-CAROTENE ZDS -2H PRONEUROSPORENE ZDS -2H PROLYCOPENE ALL-TRANS LYCOPENE CRT ISO LYCOPENE LCYE LCYB LCYE LCYB LCYB CAROTENE CAROTENE CAROTENE CAROTENE CAROTENE RING RING
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Hydroxylation of α and β-carotene (Figure 1.5) will produce the well known xanthophyll pigments zeaxanthin and lutein respectively. Violaxanthin is formed from zeaxanthin through epoxidation (Figure 1.6). This reaction sequence is reversible and de- epoxydation can convert violaxanthin back to zeaxanthin. Neoxanthin is synthesised and derived from violaxanthin (Cunningham and Gantt, 1998; Howitt and Pogson 2006).
Figure 1.5: Formation of xanthophylls through hydroxylation and addition of a keto group. Abbreviations: CHYE, ε-ring hydroxylase; CHYB, β-ring hydroxylase.
Figure 1.6: Formation of xanthophylls through hydroxylation, epoxidation and deepoxidation. Abbreviations: CHYB, β-ring hydroxylase;
VDE, violaxanthin deepoxidase; ZEP,
zeaxanthin epoxidase and NXS, neoxanthin synthase.
The study of carotenogenic enzymes remains a very difficult task and, although several crude cell-free preparations from other natural sources have been performed, pure enzymes have not been obtained and the characteristics of the enzyme-catalysed reactions have not been established (Britton, 1989). It is generally believed that carotenoid biosynthesis takes place on a multienzyme complex which is bound to, and may be an integral part of a membrane (Britton, 1989). It is however relatively easy to isolate cell-free preparations which are capable of converting mevalonic acid (MVA), isopentenyl diphosphate (IDP) or GGDP into phytoene (Britton, 1989). Phytoene synthase is considered to be peripheral to the membrane and is generally easily dissociated and
O O O OH O HO HO HO OH CAROTENE CAROTENE CRYPTOXANTHIN LUTEIN CANTHAXANTHIN ASTAXANTHIN CHYE CHYB KETOLASE HYDROXYLASE CHYB HO CHYB OH HO VDE OH HO O ZEP VDE ZEP HO O OH O NXS OH O OH HO CAROTENE CRYPTOXANTHIN ZEAXANTHIN ANTHERAXANTHIN VIOLAXANTHIN NEOXANTHIN
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solubilized. The later enzymes for desaturation, cyclization, hydroxylation and other modifications are much more difficult to deal with and are assumed to be membrane- bound. The biosynthesis of phytoene from precursors such as MVA, IDP, GGDP and PPDP has been demonstrated with crude or partially purified enzyme systems from many plants, fungal and bacterial sources (Britton, 1989). Chloroplast systems have been notoriously poor at metabolizing phytoene. In general the most active parts for carotenoid biosynthesis in higher plants are derived from chromoplasts such as in Narcissus flowers and in tomato and Capsicum fruits (Bramley, 1989). Carotenoid biosynthesis is regulated by several factors, including light (Bramley and Mackenzie, 1987). Many fungi exhibit photoregulation, typically by blue light, and either produce carotenoids only in the light such as Neurospora and Aspergillus, or show a large increase in carotenogenesis upon illumination such as Phycomyces (Rau, 1985). Photoregulation usually occurs at the level of gene expression (Bramley, 1989).