Carotene desaturation

ALBRECHT M, KLEIN A, HUGUENEY p, SANDMANN G and KUNTZ M (1995) Molecular cloning and functional expression in E. coli of a novel plant enzyme mediating -carotene desaturation , FEES Lett, 372, 199-202. 

MAYER M p, NiEVELSTEiN V and BEYER p (1992) Purification and characterisation of aNADPH-dependent oxidorednctase from chloroplasts of Narcissus-a redox mediator possibly involved in carotene desaturation . Plant Physiol Biochem, 30, 389-98. 

MORSTADT L, GRABER P, DE PASCALIS L, KLEINIG H, SPETH V and BEYER P (2002) Chemiosmotic ATP synthesis in photo synthetically inactive chromoplasts from Narcissus pseudonarcissus L. linked to a redox pathway potentially also involved in carotene desaturation , Planta, 215, 132-40. 

Beyer, R, Mayer, M., and Kleinig, K., Molecular oxygen and the state of geometric isomerism of intermediates are essential in the carotene desaturation and cyclization reactions in daffodil chromoplasts, Eur. J. Biochem. 184, 141, 1989. 

Beyer, R, Croncke, U., and Nievelstein, V., Biochemical aspects of carotene desaturation and cyclization in chromoplasts membranes from Narcissus pseudonarcissus. Pure Appl. Chem. 66, 1047, 1994. 

LUO, R., Molecular and genetic studies related to zeta-carotene desaturation and carotenoid biosynthesis in maize and rice, Ph.D. Dissertation, City University of New York, 2000. 

Direct interaction with the enzyme [Pg.191]

Some time ago we developed conditions to investigate this system. Conditions were defined for the successful solubilization of integral membrane enzymes, using CHAPS, a zwitterionic detergent, and for reconstituting these enzymes into proteoliposomes (Beyer et al. 1985, Beyer 1985). As it is possible to separate proteins in micellar solution under certain circumstances, it should also be possible to characterize individual enzymes of the sequence by use of the reconstituted system. Problems, however, arise in such a system when a reaction is mediated by more than one enzyme and it will be shown that indeed carotene desaturation proceeds by cooperation of the membrane-integral desaturases with peripheral protein factors. In addition to these problems there are two other crucial points as to the nature of the terminal electron acceptor and - as has also been found -the state of geometrical isomerism of the carotene intermediates. 

Table 2 Inhibition of phytoene- and C"carotene desaturation by J334 and Jq04 using radiolabelled phytoene and -carotene as substrates.

In the sequence of carotene desaturation are two targets for herbicide interaction the step from phytoene to -carotene and the step from -carotene to lycopene. It is assumed that two individual desaturase systems work in sequence. 

Whatever number of proteins are responsible for carotene desaturation, there is general agreement that the structural genes are nuclear rather than chloroplastidic and that the proteins are synthesized on SOS ribosomes prior to transport into the plastid. 

The desaturation of l5-cis phytoene into lycopene occurs in four stepwise dehydrogenations, yielding phytofluene, ( -carotene, neurosporene and lycopene... 

Isomerisation of 15-c/5-phytoene to the all-/ra x configuration must occur during the desaturation steps, since most desaturated carotenes are in the all-trans form. The CRTI type desaturases appear to be able to carry out this isomerisation themselves (Fraser et al, 1992 Bartley etal, 1999), but mutants of PDS/ZDS-type organisms accumulate cis isomers of unsaturated carotenes, suggesting the presence of a separate isomerase (Clough and Pattenden, 1983 Ernst and Sandmann, 1988). Three recent publications have reported the cloning of a carotene isomerase (CrtlSO) from tomato (Isaacson et al, 2002), Arabidopsis (Park et al, 2002) and Synechocystis 6803 (Breitenbach... 

Over-expression of bacterial phytoene synthase led to only modest increases in pigment accumulation (except in the case of chloroplast-contaiifing tissues). Attention turned to CrtI, one gene that might control flux through the entire four desaturation steps from phytoene to lycopene (discussed in Section 5.3.2.4). Only a modest increase in carotenoid content in tomatoes and a variety of changes in carotenoid composition including more P-carotene, accompanied by an overall decrease in total carotenoid content (no lycopene increase), resulted when CrtI was over-expressed under control of CaMV 35S. Apparently, the bacterial desaturase... 

A series of desaturation reactions convert phytoene to i -carotene and then to lycopene, the important red pigment in tomatoes. In pepper, lycopene undergoes a cyclization reaction on both ends by lycopene P-cyclase, thus producing P-carotene (Fig. 8.2) [25]. Beta-carotene is then converted to -cryptoxanthin, zeaxanthin. 

Phytoene can be converted to the carotenes by pathways indicated in part in Fig. 22-5 and Eq. 22-10. One of the first products is lycopene, the red pigment of tomatoes and watermelons, which is an all-trans compound. If 15-Z phytoene is formed, it must, at some point, be isomerized to an all-E isomer, and four additional double bonds must be introduced. The isomerization may be nonenzymatic. The double bonds are created by an oxygen-dependent desaturation, which occurs through the trans loss of hydrogen atoms. 

FIGURE 63.1 Starting with mevalonate, carotenoids are biosynthesized by a special branch of the terpenoid pathway. The first C-40 hydrocarbon unit formed is phytoene, a carotenoid with three conjugated double bonds, which then is enzymatically desaturated to successively yield (3-carotene, neurosporene, and lycopene. Other carotenoids such as (3-carotene and oxocarotenoids are produced from lycopene following cyclization and hydroxylation reactions. Thus, lycopene is a central molecule in the biosynthesis pathway of carotenoids. 

A great diversity in molecular structure is observed among herbicides which inhibit carotene biosynthesis as is exemplified by the structures of norflurazon, fluridone and difunone (shown below). Nonetheless, many of these compounds, which comprise a subset of the larger group known as bleaching herbicides, appear to inhibit the same step in the biosynthetic pathway to the carotenoids (1 ). The inhibited step is the desaturation of 15-cis phytoene to 15- cis phytofluene (Figure 1) and the build-up of phytoene in plants and in cell-free systems which have been treated with these herbicides is well documented (2-4). 

The biosynthesis of carotenoids in plants has been reviewed extensively in recent years and is only briefly described here (Britton, 1988 Bartley and Scolnik, 1994 Sandmann, 1994). The committed step to carotenoid synthesis is the formation of the first compound phytoene by the head-to-head condensation of two molecules of GGDP by phytoene synthase. Phytoene is subjected to a series of four sequential desaturation reactions, by two separate enzymes to yield lycopene, which has eleven conjugated double bonds. Lycopene is then cyclized to /3-carotene by two /3-cyclizations or to a-carotene... 

Phytoene is desaturated to neurosporene or lycopene by the photosynthetic bacteria. These are then converted to carotenes by desaturation, saturation, cyclization and aromatization, and/or to... 

During desaturation, two pathways are involved via -carotene and asymmetrical -carotene (Fig. 3). -Carotene is found in Rba. capsulatus, Rba. sphaeroides and Rvi. gelatinosus (S. Takaichi, unpublished), Chi. limicola and Pld. luteolum (Schmidt and Schiburr, 1970) and Rsb. denitrificans (Harashima and Nakada, 1983), while asymmetrical f-carotene in Rsp. rubrum (Davies, 1970), Rpi. globiformis (Schmidt and Liaaen-Jensen, 1973), Rmi. vannielii (Britton et al., 1975), Bla. viridis, Chi. tepidum and Cfl. aurantiacus (S. Takaichi, unpublished) and Erb. longus (Takaichi et al., 1990). The two pathways via f-carotene and asymmetrical... 

Norfluorazon is an often-used inhibitor of carotenoid biosynthesis, interfering with the step ofphytoene desaturation (Sandmann, 1994). Norflurazon treatment of mustard seedlings showed that PS II assembly is more sensitive to reduced levels of carotenoids than PS I assembly (Markgraf and Oelmiiller, 1991). The assembly of D1 into the PS II reaction center appears to be dependent on /3-carotene. Upon treatment of Chlamydomonas reinhardtii with phytoene desaturase inhibitors, D1 degraded during photoinhibition caimot be replaced (Trebst and Depka, 1997). 

Subsequent desaturation reactions lengthen the conjugated double-bond system to produce neurosporene or lycopene. Two completely unrelated types of phytoene desaturases exist. The enzyme found in bacteria (except cyanobacteria) and in fungi catalyzes the entire four-step desaturation process of phytoene to lycopene (Schmidt-Dannert 2000). The plant-type phytoene desaturase from cyanobacteria, algae, and plants carries out a two-step desaturation reaction with different -carotene stereoisomers as reaction products (Britton et al. 1998, Schmidt-Dannert 2000). 

Several phytoene desaturases are known that differ in the number of desaturation steps and in their structures. Among them, the bacteriaPfungal type is encoded by crti, and the cyanobacterial/algal/plant type is encoded by crtP or pds. Phytoene desaturase converts phytoene to -carotene with phytofluene as an intermediate. The reaction is stimulated by NAD, NADP, and oxygen. 

Carotene desaturase catalyzes the final two desaturation steps to lycopene by introducing two double bonds at positions 7,8 of -carotene and 7, 8 of neurosporene. From cyanobacteria, two structurally unrelated genes for -carotene desaturase have been cloned. One crtQ (formerly called zds) from Anabaena is related to the bacterial crtI gene, whereas the second crtQb (also called crtQ-2) from Synechocystis is quite similar to crtP. 

A cell-free system from photosynthetically active species has to be developed that is able to produce substantial amounts of intermediates and desaturated carotenes. Noteworthy a recent report using isolated chromoplasts from daffodil flowers. Narcissus pseudonarcissus > and isopentenyl pyrophosphate as substrate noted increased levels of phytoene and geranylgeraniol vs. control in the presence of high concentration (50 j,M) of SAN 6706 (27). These cell-free assays should be investigated further with regard to possible different sensitivity against inhibitors because of the species used. 

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