Phytoene biosynthesis

The last step in the biosynthesis of phytoene is the head-to-head condensation of two molecules of GGPP (Fig. 8). In most respects, this reaction is analogous to the condensation of two molecules of famesyl pyrophosphate to form squalene in the sterol biosynthetic pathway. Studies on the latter reaction have provided a great deal of insight into the mechanism of phytoene biosynthesis and are included in the discussion below where relevant. Both reactions involve the formation of a cyclopropylcarbinyl pyrophosphate intermediate. In the biosynthesis of phytoene this compound is pre-phytoene pyrophosphate, the C40 analogue of presqualene pyrophosphate. A proposed mechanism for the formation of prephytoene pyrophosphate is shown in Fig. 9 (Beytia and Porter, 1976). Prephytoene pyrophosphate has been isolated from a Mycobacterium preparation and synthesized chemically. Both the natural and synthetic compounds have been converted to phytoene by a cell-fiee system from Mycobacterium (Altman et al., 1972). Prephytoene pyrophosphate was also formed when GGPP was incubated with yeast squalene synthetase (Qureshi et al., 1972, 1S>73), but in the pres-... 

Clearly, the control of gene expression at the transcriptional level is a key regulatory mechanism controlling carotenogenesis in vivo. However, post-transcriptional regulation of carotenoid biosynthesis enzymes has been found in chromoplasts of the daffodil. The enzymes phytoene synthase (PSY) and phytoene desaturase (PDS) are inactive in the soluble fraction of the plastid, but are active when membrane-bound (Al-Babili et al, 1996 Schledz et al, 1996). The presence of inactive proteins indicates that a post-translational regulation mechanism is present and is linked to the redox state of the membrane-bound electron acceptors. In addition, substrate specificity of the P- and e-lycopene cyclases may control the proportions of the p, P and P, e carotenoids in plants (Cunningham et al, 1996). 

The carotenoid pathway may also be regulated by feedback inhibition from the end products. Inhibition of lycopene cyclisation in leaves of tomato causes increase in the expression of Pds and Psy-1 (Giuliano et al, 1993 Corona et al, 1996). This hypothesis is supported by other studies using carotenoid biosynthesis inhibitors where treated photosynthetic tissues accumulated higher concentrations of carotenoids than untreated tissues (reviewed by Bramley, 1993). The mechanism of this regulation is unknown. A contrary view, however, comes from studies on the phytoene-accumulating immutans mutant of Arabidopsis, where there is no feedback inhibition of phytoene desaturase gene expression (Wetzel and Rodermel, 1998). 

POTRYKUS I (1997) Transgenic rice Oryza sativa) endosperm expressing daffodil Narcissuspseudonarcissus) phytoene synthase accumulates phytoene, a key intermediate of provitamin A biosynthesis , Plant J, 11, 1071-78. 

Dogbo, O. et al., Carotenoid biosynthesis isolation and characterization of a bifunctional enzyme catalyzing the synthesis of phytoene, Proc. Natl. Acad. Sci. USA 85, 7054, 1988. 

Powls, R. and Britton, G., The roles of isomers of phytoene, phytofluene and zeta-carotene in carotenoid biosynthesis by a mutant strain of Scenedesmus obliquus. Arch. Microbiol. 115, 175, 1977. 

The molecular target site of triketone herbicides is the enzyme -hydroxyphenylpyruvate dioxygenase (HPPD). Inhibition of this enzyme disrupts the biosynthesis of carotenoids and causes a bleaching (loss of chlorophyll) effect on the foliage similar to that observed with inhibitors ofphytoene desaturase (e.g. norflurazon). However, the mechanism of action of HPPD inhibitors is different. Inhibtion of HPPD stops the synthesis of homogen tisate (HGA), which is a key precursor of the 8 different tocochromanols (tocopherols and tocotrienols) and prenyl quinones. In the absence of prenylquinone plastoquinone, phytoene desaturase activity is interrupted. The bleaching of the green tissues ensues as if these compounds inhibited phytoene desaturase. 

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. 

Enzyme Systems. Carotenoid biosynthesis by crude cell-free preparations from Halobacterium cutirubrum 0-carotene), Phycomyces blakesleeanus mutants (/8-carotene), and a Neurospora crassa mutant (phytoene) has been demonstrated. Detailed studies of carotenogenic enzymes from tomato fruit... 

Light is a major regulatory influence on carotenoid synthesis in many plant and microbial systems. A review of this photoregulation has been published. Other papers report the photoinduction of the biosynthesis of phytoene and other carotenoids in strains of Neurospora crassa. " ... 

Carotenoids produced in plants are used as colorants in foods and aiflmal feeds and can also have an antioxidant function. Production of phytoene synthase is the first committed step towards carotenoid biosynthesis in plants. When phytoene synthase is produced in B. napus, a 50-fold increase in carotenoid expression results. Therefore Brassica... 

The enzyme p-hydroxyphenylpyruvate dioxygenase is involved in the conversion of p-hydroxyphenylpyruvate into homogentisate, a key step in plastoquinone biosynthesis. Inhibition of this enzyme has an indirect effect on carotenoid biosynthesis as plastoquinone is a co-factor of the enzyme phytoene desaturase. The new maize herbicide isoxaflutole and the triketone herbicides such as sulcotrione (Figure 2.7), inhibit p-hydroxyphenylpyruvate dioxygenase and this leads to the onset of bleaching in susceptible weeds and ultimately plant death.4... 

F, Bleaching inhibition of carotenoid biosynthesis at the phytoene desaturase step (PDS) Pyridazinones Nicotinanilides Others 12... 

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. 

Inhibitors of carotenoid synthesis also lead to chlorophyll destruction by destabilizing the photosynthetic apparatus. Total carotenoid content decreased with increased (-)-usnic concentration (Fig. 1.4). Carotenoid biosynthesis can be interrupted by inhibiting the enzyme phytoene desaturase that converts phytoene to carotenes or by inhibiting the enzyme HPPD responsible for plastoquinone (required for phytoene desaturase activity) synthesis.14 Usnic acid possesses some of the structural features of the triketone HPPD inhibitors, such as sulcotrione (Fig. 1.1C).8 (-)-Usnic acid had a strong inhibitory activity on HPPD, with an apparent IC50 of 70 nM, surpassing the activity obtained with the commercial herbicide sulcotrione (Fig. 1.5). 

The amount of carotenoids formed by biosynthesis was quantified by the accumulation of the colourless carotenoid phytoene in the presence of the inhibitor, norflurazon. When applied, substantial amounts of this... 

Most of the compounds cited in this introductory section are produced in metabolic processes where the cyclopropane-containing metabolite appears to be the stable end product or secondary product with as yet unobvious metabolic function. However, this is not the case in at least two types of systems, in which cyclopropyl species are key and necessary intermediate structures in high flux metabolic pathways. The first example is the squalene (76) and phytoene (88) biosynthesis where presqualene pyrophosphate (77) and prephytoene pyrophosphate (89) are obligate cyclopropanoid intermediates in the net head-to-head condensations of two farnesyl pyrophosphate (73) or two geranylgeranyl pyrophosphate (66) molecules respectively. The second example is in plant hormone metabolism where C(3) and C(4) of the amino acid methionine are excised as the simple hormone ethylene via intermediacy of 1-aminocyclopropane-l-carboxylic acid (9). Both examples will be discussed in detail in the Section II. 

First, we will take up cyclopropyl group formation by the rearrangement of carbon skeletons via cationic intermediates encountered in various mono- and sesquiterpenes, and also examine the illudin biosynthesis where contraction of a cyclobutyl cation to a cyclopropane has been invoked. We will then discuss the head-to-head condensation of isoprenoid alcohols at the C15 or C20 level to generate the cyclopropyl intermediates, presqualene pyrophosphate and prephytoene pyrophosphate, on the way to the C30 and C40 polyene hydrocarbons, squalene and phytoene respectively. Conversion of 2,3-oxidosqualene via common intermediate protosterol cation to cycloartenol or lanosterol represents an important pathway in which the angular methyl group participates in the three-membered ring formation. The cyclopropanation outcome of this process has been carefully studied. 

In this section we analyze information about metabolic cleavage or breakdown of cyclopropane rings in three instances the biosynthesis of irregular monoterpenes, the ringopening of cycloartenol (20) derivatives, and the metabolic opening of 1-aminocyclopropane-1-carboxylic acid (ACPC) (9) by two quite distinct fragmentation routes. We will not explicitly discuss the processing of presqualene pyrophosphate (77) and prephytoene pyrophosphate (89) to squalene (76) and phytoene (88) respectively, since those transformations have already been dealt with in Section II. 

Interest in the action of various chemicals on carotenoid biosynthesis has been maintained in Phycomyces blakesleeanus and its mutants, diphenylamine caused increased levels of phytoene and phytofluene and reduced levels of coloured carotenes, whereas dimethyl sulphoxide reduced both types.The drug AMO 1618 increased the levels of all types of carotenoids in all strains, probably by preventing cyclization of GGPP and so increasing the amount of this precursor available for dimerization. The Et2NCH2 group in the amines (106)—(108) was... 

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

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