Prephytoene pyrophosphate

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. 

The intermediacy of prephytoene pyrophosphate (89) was first shown by Altman and coworkers who identified this compound as a product of the incubation of geranylgeranyl pyrophosphate (66) with an extract of photoinduced Mycobacterium sp. The direct incorporation of geranylgeranyl pyrophosphate (66) into phytoene (88) has been observed in various systems including a soluble tomato plastid enzyme systemchloroplasts isolated from Phaseolus vulgaris cell-free extracts of P. blakesleeanus and... 

Formation of prephytoene pyrophosphate (89) might proceed, as proposed by Altman and coworkers , via a Mg -assisted solvolysis of the pyrophosphate from C(l) of the prenyl donor with concomitant carbon-carbon bond formation between C(l) and C(2) as well as C(3) of the prenyl acceptor, or other mechanisms analogous to those proposed for presqualene pyrophosphate (77) formation. Although numerous chemical model studies have been reported, there is no conclusive evidence for enzymatic use of any of these mechanisms so far. 

Samples of (IK, 2R, 3R) and (IS, 25, 35) prephytoene alcohols have been chemically synthesized and only the (IK, 2K, 3K) enantiomer (91), as the pyrophosphate ester, is biologically active The absolute configuration of prephytoene pyrophosphate (89) is thus identical to that found for presqualene pyrophosphate (77) and the detailed stereochemistry of the formation of these compounds is probably identical. 

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. 

The crucial intermediacy of cyclopropylcarbinyl species in the biological synthesis of hundreds (thousands) of steroids, carotenoids, retinoids and derivatives is exemplified by the C30 presqualene pyrophosphate (77) and the C40 prephytoene pyrophosphate (89). In the biosynthetic construction of the key C(l)-C(l) carbon-carbon in head-to-head joining of Cl 5 or C20 alkyl alcohol pyrophosphate esters, the cyclopropylcarbinyl strategy via a formal insertion of C(l) of one monomer into the C(2)-C(3) double bond of the second monomer appears to be the central mechanistic solution in the biochemical inventory. The cyclopropylcarbinyl pyrophosphate forms as obligate intermediate whether the final... 

Stereochemistry. It is well known that formation of the first C40 hydrocarbon in carotenoid biosynthesis proceeds via an intermediate, prephytoene pyrophosphate. It has now been shown that only the pyrophosphate of the (-1-)-(l/ ,2/ ,3i )-isomer of prephytoene alcohol is utilized for carotene production by Phycomyces blakesleeanus. Detailed mechanisms for the formation of (l/ ,2/ ,3/ )-prephytoene pyrophosphate (178) from geranylgeranyl pyrophosphate (177) have been proposed (Scheme 4). - ... 

Two albino mutants of Neurospora crassa have been isolated in which carotenoid biosynthesis is blocked between prephytoene pyrophosphate and phytoene. ... 

GGPP geranylgeranyl pyrophosphate IPP isopentenyl pyrophosphate PPPP prephytoene pyrophosphate. 

Formation of Geranylgeranyl Pyrophosphate Formation of Prephytoene Pyrophosphate Formation of Phytoenes Acyclic Carotenoids Alicyclic Carotenoids Oxygenated Carotenoids Site of Synthesis Chemosystematic Studies Carotenoids in Algae Cartenoids in Fungi Biological Activity... 

The first step in the formation of prephytoene pyrophosphate (10) and phytoene (8) is the prenyl-transfer step, during which C(T) of one of the allylic substrates is bonded to the C(2)-C(3) double bond of the other to produce a cyclopropylcarbinyl pyrophosphate with a CT-2-3 stmcture (Fig. 26.2). In this manner, geranylgeranyl pyrophosphate (9) yields prephytoene pyrophosphate (10) (Poulter, 1990). 

In some instances, the ability to produce prephytoene pyrophosphate (10) is closely linked to the production of phytoene (8) and appears to involve one enzyme with two activities (Britton, 1993 Dogbo et al., 1988). With an enzyme isolated from Capsicum chromoplast preparations, biosynthesis precedes via condensation of two molecules of geranylgeranyl pyrophosphate (9) directly to phytoene (8) (Dogbo et al., 1988). The enzyme phytoene synthase has been purified to homogeneity phytoene synthase from this source is a monomer wirti a molecular weight of 47,500 (Dogbo et al., 1988). 

Although the enzyme from Capsicum has both types of activity, prephytoene pyrophosphate is an isolable product in other situations. The conversion of GGPP (9) to prephytoene pyrophosphate (10) has been demonstrated with a soluble tomato plastid enzyme system and chloroplasts isolated from Phaseolus vulgaris by nonaqueous methods (Spurgeon and Porter, 1983). The only cofactor that appears to be required is Mg + or Mn. There is no requirement for a reduced pyridine nucleotide as exists for the formation of squalene. Prephytoene pyrophosphate (10) appears to be the only isolable intermediate involved in the process. This compound has the same absolute stereochemistiy as presqualene pyrophosphate. 

Fig. 26.2. Proposed mechanism for the conversion of GGPP to prephytoene pyrophosphate.

Fig. 26.3. Proposed mechanism for the conversion of prephytoene pyrophosphate to lycopersene, Z-phytoene, and -phytoene (Poulter, 1990 adapted and used with permission of the copyright owner, the American Chemical Society, Washington, DC).

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-... 

Fig. 9. A proposed mechanism for the conversion of GGPP to prephytoene pyrophosphate. (Reproduced by permission of the authors and the publisher from Beytia el al., 1973.)...

Most of the natural carotenoid pigments are tetraterpenes, being formed from two C20 geranylgeranyl pyrophosphate units through phytoene 4.122). The mechanism of phytoene formation has much in common with squalene biosynthesis. In particular, head-on fusion of the two C20 units occurs via prephytoene pyrophosphate 4.120), analogous to presqualene pyrophosphate 4.48) formation from two... 

Prephytoene pyrophosphate as an intermediate of the sequence was established by Gregornis and Rilling (ref. 13). Porter s group investigated the phytoene synthase complex which was obtained from a relatively crude chromoplast preparation from tomato fruits and from an acetone powder prepared thereof. The complex was partially purified and a molecular mass of about 166 000 Da was determined (ref. 14). The dissociation of an isomerase and prenyltransferase activity was achieved (molecular masses about 34 000 Da and 64 000 Da, respectively) (ref. 15). Furthermore, a strict dependency on Mn was established which is known for preny1transferases from other sources. A severalfold stimulation of phytoene formation by ATP was observed, although the hydrolysis of ATP is not required within this enzymatic sequence (ref. 16). 

The first reaction of isoprenoid biosynthesis that is specific for the carotenoids is the head-to-head condensation of two molecules of GGPP into phytoene, via the intermediate prephytoene pyrophosphate (PPPP Figure 4.2). The conversion of PPPP into phytoene involves the loss of a hydrogen atom to yield, in plants, the 15-Z isomer. This reaction has been demonstrated in several cell extracts, such as tomato plastids," chloroplasts of Triticum sativum, and plastids of Capsicum annuum," The alternative, all-F isomer of phytoene is not found in higher plants. 

The biosynthesis of phytoene (296c), the parent of the carotene family, also proceeds via a cyclopropane intermediate referred to as prephytoene pyrophosphate (113c) (772, 379). Phytoene is derived from (113c) by the same rearrangement sequence, the termination step being proton elimination instead of hydride transfer. 

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