Phytoene synthesis

A further consideration in the choice of gene relates to those that are members of a gene family. Only one member of such a family may be involved in carotenogenesis in a particular tissue. A good example of this is Psy-1 and-2 of tomato. PSY-1 is responsible for phytoene synthesis in ripening fruit (Fraser et al, 1999) whereas PSY-2 is not functional in chromoplasts, even if the protein is produced. 

Presqualene pyrophosphate (32), a compound whose structure has caused considerable controversy in the past, has been isolated from intact rat liver and a yeast microsomal system. Previously, (32) had been detected only in systems which have been starved of NADH and hence the new findings demonstrate that (32) is not an artefact. Despite earlier evidence that lycopersene is a precursor of phytoene, a recent stereochemical analysis of phytoene synthesis makes this appear to be unlikely, and a mechanism has been proposed for the synthesis of cis- and tmnj -phytoene directly from pre-phytoene pyrophosphate (33) (Scheme 7). This mechanism is similar to one proposed for squalene synthesis. ... 

All carotenoids are derived from the isoprenoid or terpenoid pathway. From prenyl diphosphates of different chain lengths, specific routes branch off into various terpenoid end products. The prenyl diphosphates are formed by different prenyl transferases after isomerization of IPP to DMAPP by successive T-4 condensations with IPP molecules. Condensation of one molecule of dimethylallyl diphosphate (DMADP) and three molecules of isopentyl diphosphate (IDP) produces the diter-pene geranylgeranyl diphosphate (GGDP) that forms one-half of all C40 carotenoids. The head-to-head condensation of two GGDP molecules results in the first colorless carotenoid, phytoene. Phytoene synthesis is the first committed step in C40 carotenoid biosynthesis (Britton et al. 1998, Sandmann 2001). 

Phytoene synthase is encoded by the closely related bacterial crtB and eukaryotic psy genes. The phytoene formed in the enzymatic reaction was present in both a 15-cw and all-trans isomeric configuration. The essential cofactors required were adenosine triphosphate (ATP) in combinations with either Mn + or Mg +. Phytoene synthesis was inhibited by phosphate ions and squalestatin. 

THE ROLE OF ISOPENTENYL-DIPHOSPHATEid-ISOMERASE IN PHYTOENE SYNTHESIS OF DAFFODIL CHROMOPLASTS... 

At the moment our work concentrates on the characterisation of this branch point of monoterpene synthesis and phytoene synthesis, respectively. Both ways can be activated by different means. Phytoene synthesis is stimulated by 3 mM ATP and liposomes whereas optimal monoterpene synthesis occurs in the absence of ATP and liposomes. 

Studies in the 1960s and 1970s on the cofactor requirements for phytoene synthesis by cell extracts of higher plants produced conflicting results (see Ref. 66). More definitive experiments over the past few years have clarified some of these anomalies. In particular, the enzyme has been purified to homogeneity from the chromoplast stroma of Capsicum annuum, to yield a single protein, 47,500, with an absolute requirement for Mn ", but no other cofactors. This enzyme catalyzes a two-step, kinetically... 

A second class of herbicides primarily affects ( -carotene desaturase. These herbicides are apparent feedback inhibitors of PD as well. This class of compounds includes dihydropyrones like LS 80707 [90936-96-2] (56) and 6-methylpyridines (57,58). The third class consists of the ben2oylcyclohexane-diones, eg, 2-(4-chloro-2-nitroben2oyl)-5,5-dimethyl-cyclohexane-I,3-dione. This class of atypical bleaching herbicides induces phytoene accumulation when appHed either pre- or post-emergence. However, it does not inhibit phytoene desaturase activity in vitro (59). Amitrole also has been considered a bleaching herbicide, though its main mode of action is inhibition of amino acid synthesis. 

FRASER P D, KIANO I W, TRUESDALE M R, SCHUCH W and BRAMLEY P M (1999) PhytOene synthase-2 enzymes activity in tomato does not contribute to carotenoid synthesis in ripening fruit . Plant Mol Biol, 40, 687-98. 

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. 

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. 

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. " ... 

Phytoene desaturase bleaching herbicides. Bleaching herbicides inhibit the synthesis of carotenoids in plants [30,31], A number of phytoene desaturase herbicides such as norflurazon (Solicam , Zorial ), flurochloridone (Racer ), fluri-done (Sonar ), and diflufenican (Fenican , Legacy ) have been commercialized. 

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). 

Stereochemistry.—Geometrical Isomerism. Details of the synthesis and the spectroscopic and stereomutation properties of the a -trans- and 15-cw-isomers of phytoene [7,8,ll,12,7, 8, ir,12 -octahydro-i/f,i/r-carotene (23)] have been... 

Figure 1. Synthesis of Carotenes from Phytoene. (Reproduced...

Beflubutamid is a newly described carotenoid synthesis inhibitor, inhibiting phytoene desaturase. It has a very low toxicity to animals but is very toxic to algae and plants. It is not mutagenic or teratogenic in standard tests. 

It can be established from these investigations that unlike triazines and diuron, norflurazon damages the photosynthetic system of the plants. The main action is the blocking of carotenoid synthesis, as a result of which carotenoid precursors (phytoene and phytofluene) are accumulated because of the inhibition of dehydrogenation reactions. 

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 (150), phytofluene (151), -carotene (152) and neurosporene (153), the biosynthetic precursors of lycopene (1), also contain the y-end group. They differ from lycopene (1) in the degree of saturation of the 7,8, 7, 8, 11,12 and/or IT,12 double bond(s). Different building blocks as starting material have therefore been used for the synthesis of these compounds. 

For the synthesis of phytoene (150) and phytofluene (151) the C20 + C20 = C40 strategy has been applied [85,86], For the preparation of phytoene (150), trans-geranyllinalool (165) was treated with phosphorus tribromide to give geranylgeranyl bromide (166) which was reacted with triphenylphosphine to give the C2o-phosphonium salt 167. The C2o-aldehyde 168 was prepared by treatment of 166 with KOH and 2-nitropropane. The Wittig reaction of 167 and 168 with PrLi gave a mixture of (E/Z)-isomers of phytoene (150) in an overall yield of 6% referred to 165 (Scheme 38). 

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