Carotenoids desaturation

Carotenoid desaturation proceeds in stages, starting with phytoene, a colorless C40 terpenoid, which is formed by the condensation of two molecules of ger-anylgeranyl pjrrophosphate through the action of the enzyme, ph3rtoene synthase (PSY). The general scheme for phytoene desaturation, first proposed by Porter and Lincoln in 1950 before the structures were known for the individual carotenoids, is very close to the scheme depicted in Figure 1 (7,8). The Porter... 

Pig.l. The pathway of carotenoid desaturation. Carotenoids are shown in the trans form to save space, and colors are only approximate. Short arrows in the pathway denote dehydrogenation locations. Notice that R. rubrum has a slightly different sequence. Arrows to the left indicate starting and stopping points of the desaturation sequence for each t5rpe of desaturase named, including a mutant 114 (6). 

Desaturation and Isomerization to Coeored Carotenoids Biosynthesis of Lycopene... 

The carotenoid isomerase (CRTISO) was the first isomerase associated with the desaturation steps and named at a time when Z-ISO was unknown to exist ise.ws.ieo.iei (and reviewed in references ). In vitro analysis of substrate conversion " and transcript profiling in planta associated CRTISO with the desaturation steps. Isaacson demonstrated that CRTISO is specific for the 7,9 or 7,9- cis bond configuration and is not involved in the isomerization of the l5-l5-cis double bond to the trans conformation. As recently shown, Z-ISO is required for isomerization of the 15-15 cis double bond of phytoene produced in dark-grown tissues as well as in stressed photosynthetic tissues. Therefore, desaturation of phytoene to lycopene involves a two-step desaturation by PDS, followed l5-cis isomerization by Z-ISO, and then each pair of double bonds introduced by ZDS is followed by CRT-ISO-mediated isomerization of the resulting conjugated double bond pair. 

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... 

Fortuitously, the bacterial gene product, CRTI, produces di -trans carotenoids and satisfies the stereo-chemical specificity of LYC B for all-trani substrates while also catalyzing the four desaturation steps from phytoene to lycopene. Nevertheless, over-expression of Crtl has been shown to have only a modest effect (two- to fourfold increases in tomatoes and carrots) in increasing flux through the pathway and some unexpected pleiotropic influences on activities upstream and downstream of the desaturations (reviewed by Fraser and Bramley and Giuliano °). 

Matthews, P.D., Luo, R., and Wurtzel, E.T., Maize phytoene desaturase and zetacar-otene desaturase catalyze a poly-Z desaturation pathway implications for genetic engineering of carotenoid content among cereal crops, J. Exp. Botany 54, 2215, 2003. 

Biosynthesis and Metabolism.—Pathways and Reactions. Two reviews of carotenoid biosynthesis discuss, respectively, the early steps and the later reactions." The former paper deals with the mechanism of formation of phytoene and the series of desaturation reactions by which phytoene is converted into lycopene, and also describes in detail the biosynthesis of bacterial C30 carotenoids. The second paper" presents details of the mechanism and stereochemistry of cyclization and the other reactions that involve the carotenoid C-1 —C-2 double bond and the later modifications, especially the introduction of oxygen functions. 

Desaturation takes place in a stepwise fashion, and many intermediate compounds with fewer double bonds are known (Eq. 22-10).118/121-123 The enzymes required have not been characterized well until recently. Plant enzymes are present in small amounts, and isolation has been difficult. However, the genes for carotenoid biosynthesis in such bacteria as the purple photosynthetic RhodobacterRhodospirillum,125 and Rubrivarax,126 the cyanobacterium Synechococcus,127 and the nonphotosynthetic Erwinia44/118 have been cloned, sequenced, and used to produce enzymes in quantities that can be studied. Matching genes from higher plants have also been cloned and expressed in bacteria.123... 

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. 

Two phytoene desaturase herbicides have been introduced since 2000 picolina-fen (Pico ) [182], introduced in 2001 by BASF, and beflubutamid [183], introduced in 2003 by Ube Industries. The primary mode of action of picolinafen and beflutamid is interference of carotenoid biosynthesis at the phytoene desaturation level, causing bleaching of the plant affected. As in previously developed phytoene desaturase herbicides, a meta-substituted trifluoromethylphenyl group is key for activity in this class of herbicides, pointing to the need for a lipophilic and electron-withdrawing group at this position of the molecule. 

Carotenoid Biosynthesis Destruction of Pigments (isoprenoid formation, desaturation, cylase, a hydroxylatlon) T... 

Inhibition and Regulation. There have been several reports on the effects of desaturation and cyclization " inhibitors on carotenoid biosynthesis in various micro-organisms and plant tissues. 

In the photosynthetic bacteria Rhodomicrobium vannielii, which normally contains acyclic carotenoids with tertiary hydroxy- and methoxy-groups at C-1 and C-T, phytoene only accumulated when diphenylamine was present, but the occurrence of hydroxy-derivatives of phytofluene, 7,8,11,12-tetrahydrolycopene, neurosporene, and lycopene in the presence of the inhibitor indicated that hydroxylation could take place at any level of desaturation although only the more desaturated half of the molecule was so substituted. 

FIGURE 4.4 Scheme for the stepwise desaturation of phytoene to lycopene in carotenoid biosynthesis. (Goodwin, 1980. With permission.)... 

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... 

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). 

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