Phytoene desaturation

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. 

MAYER, M. P., BEYER, P., KLEINIG, K., Quinone compounds are able to replace molecular oxygen as tenninal electron acceptor in phytoene desaturation in chromoplasts of Narcissus pseudonarcissus L., Eur. J. Biochem. 1990, 191, 359-363. 

Most commercial so-called bleaching herbicides inhibit the synthesis of carotenoids by interfering with carotenoid biosynthesis at the level of phytoene desatur-ase [170, 171, 172]. Errzyme kinetics with several inhibitors have revealed a reversible binding to the en2yme and non-competitive inhibition [173],... 

In order to exclude the possibility that inhibition of phytoene desaturation is caused by indirect or regulatory mechanisms, direct interaction of substituted phenylthiazolines with phytoene desaturase was demonstrated by in vitro studies. As a result, with increasing concentrations, more phytoene is retained which means that less phytoene was desaturated. Therefore, less 3-carotene is formed as the end product of this in vitro biosynthetic chain. Interaction of phenylthiazolines is very similar to inhibition of this enzyme by flurtamone. For the latter herbicide, it has been shown to be a reversible non-competitive inhibitor of phytoene desaturase (P). 

In higher plants, fimgi, and some bacteria the predominant isomer of phytoene is the 15-cis compound (Section V,B,2). However, the more unsaturated carotenes in these same organisms are the all-trans isomers. This means that the pathway of phytoene desaturation in these organisms also involves an isomerization reaction. This isomerization occurs in an early stage of the pathway of phytoene desaturation, but the exact step at which it occurs may not be the same in all cases. In higher plants only traces of trans-... 

Although the pathway of phytoene desaturation is well established, little is known concerning the enzymes involved. The enzyme system from tomato fruit plastids which converts phytoene to lycopene is extractable with phosphate buffer after preparation of an acetone powder. Whether this enzyme system consists of individual enzymes or an enzyme complex is not known. It is clear, though, that an isomerase is also involved, as well as the enzymes that bring about the desaturation reactions. There is evidence for a multienzyme complex inP. blakesleeanus (Aragon et al., 1976). In this organism the... 

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... 

Historical Evidence for the Porter-Lincoln Pathway of Phytoene Desaturation... 

A great deal of evidence for the Porter-Lincoln pathway of phytoene desaturation came with the advent of inhibitors of various steps of carotenoid biosynthesis, the discovery of carotenoid mutants in various organisms, and in vitro biosynthesis... 

The desaturation of C carotene, on the other hand, was not influenced by these compounds, even when administered in very high concentrations (100 to 1000 times the concentration for phytoene desaturation). 

Fig. 2 Lineweaver-Burk plot of the inhibition of phytoene desaturation with Norflurazone.

Table 1 I5Q values for the inhibition of phytoene desaturation (phytofluene and Carotene formation) in the order of the effectivity of the compounds tested. 

To date, there has been no report of the purification of a protein capable of metabolizing phytoene to unsaturated carotenes, and so the only information available on the enzymology of phytoene desaturation in plants has been derived from studies of crude preparations, typically from chromoplasts. 

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... 

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. 

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. 

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. 

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. 

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). 

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