Phytoene to Lycopene

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 gelatinosus (S. Takaichi, unpublished), Chi.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 aurantiacus (S. Takaichi, unpublished) and Erb.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 

Usually the final product of the phytoene desaturase is lycopene, while in Rhodobacter, the final product is neurosporene. The deduced amino acid sequences of the crtl gene from Rhodobacter (final product, neurosporene), and Erb. longus and Erwinia (lycopene) show significant similarity, but the final products are different (Sandmann, 1994 Armstrong, 1995 Matsumura et al., 1997). The mechanism for recognition of the position on carotenoid by the phytoene desaturase is still unknown that is, how the intermediate of f-carotene or asymmetrical f-carotene and the final product of neurosporene or lycopene are controlled. 

---

Besides the capacity of CRTI to introduce all four double bonds in the conversion of phytoene to lycopene, the enzyme produces different geometric isomers than does PDS/ZDS (see graphic, side-by-side comparison in Fraser and Bramley ). CRTI produces all-trans isomers. Studies that have examined the function of the paired plant desaturases acting together, from Arabidopsis, and from maize and from... 

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 °). 

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

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). 

Fig. 11. The pathway of conversion of phytoene to lycopene. In this pathway trans structures are shown for all compounds. In plants the first two compounds in this pathway (phytoene and phytofluene) have a central 15,15 -cis double bond. An isomerization reaction converts cis-phytofluene to trans-phytolluene in plants.

The hydrogen atoms lost in the desaturation of phytoene to lycopene arise from the C-2 and C-5 positions of MV A (Fig. 12). The atoms lost are the 5-pro-R and the 2-pro-S hydrogen atoms in each case (Britton, 1976a), and thus each desaturation step involves a trans elimination of hydrogen atoms. 

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... 

Fig. 106.1 Caiotenogcaiesis pathway leading to conversion of phytoene to lycopene and fhnctionally confirmed enzymes involved in this pathway. Oxygenic phototrophs require three enzymes (CrtP, CrtQ, and CrtH), whereas anoxygenic phototrophs, bacteria, and fungi use only Crtl. See the text and Table 106.3 for precise explanations...

In the case of carotenoids, the main precursor phytoene is generated by action of phytoene synthase, strictly dependent upon Mn " enzyme, on geranylgeranyl pyrophosphate. Then, the desaturation of phytoene to lycopene involves four steps (Fig. 135.3) that are catalyzed by phytoene desaturase and -carotene desaturase. 

The Stereochemistry of Phytoene.—Naturally occurring phytoene has been reported as the 15-cis-isomer of (146) this raises the interesting question as to where in the pathway from phytoene to lycopene does the central double bond revert to the trans-configuration. The recent demonstration that phytoene from Flavobacterium dehydrogenans ° and Mucor hiemalis was... 

Figure 4.3. The desaturation sequence from phytoene to lycopene in higher plants. For convenience, all the structures are shown in the all- configuration (see Section 4.4.1.1).

---