Prephytoene

Phytoene synthase [EC 2.5.1.32] (also known as gera-nylgeranyl-diphosphate geranylgeranyltransferase and prephytoene-diphosphate synthase) catalyzes the reaction of two geranylgeranyl diphosphate to produce pyrophosphate (or, diphosphate) and prephytoene diphosphate. Isopentenyl pyrophosphate isomerase [EC 5.3.3.2] catalyzes the interconversion of isopentenyl diphosphate and dimethylallyl diphosphate. See also Geranylgeranyl Diphosphate Geranylgeranyltransferase... 

Phytoene (Fig. 22-5) is apparently formed from geranylgeranyl-PP via prephytoene-Pf whose structure is entirely analogous to that of presqualene-pp 44,117 However, no reduction by NADH is required (Eq. 22-8). It is known that the 5-pro-R hydrogen atoms of mevalonate are retained in the phytoene as indicated by a shaded box in Eq. 22-8. Elimination of the other (pro-S) hydrogen yields 15,15 -Z phytoene (a s-phytoene), while elimination of the pro-R hydrogen yields all-E (trans) phytoene. Higher plants and fungi form mostly a s-phytoene, but some bacteria produce the all-E isomer.118... 

Full details have been given28 of the previously reported29 synthesis of ( )-prephytoene alcohol (47) by a route involving condensation of the Qo sulphone (48) with the C20 ester (49). Lycopersene 1,2-epoxide [1,2-epoxy-... 

The full details of the synthesis and resolution of presqualene and prephytoene alcohols and the stereochemistry of the non-oxidative cyclization of squalene to tetrahymanol by Tetrahymena pyriformis have appeared (see Vol. 9, p. 187). 

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... 

The resolution of synthetic presqualene and prephytoene alcohols via their etienic acid derivatives has been reported. This work confirmed that the active (-f-)-enantiomers in both series have the same absolute configuration [(li , 2/ , 3/ )]. It has been established, by use of Hn.m.r., that the proton (deuteron) introduced at C-3 during the cyclization of squalene to tetrahymanol by Tetrahymena pyriformis has the 3/8 configuration. Both antipodes of the trimethyldecalol (13) have been shown to be effective inhibitors of cholesterol biosynthesis in rat liver enzyme preparations and cultured mammalian cells. The accumulation of squalene 2,3-oxide and squalene 2,3 22,23-dioxide in the treated systems indicates that inhibition occurs at the cyclization stage. The inhibitor is metabolized to the diol (14). The results of other sterol inhibition... 

Acyclic Carotenoids. The etienate esters prepared by treatment of ( )-prephy-toene alcohol with 3/3-acetoxy-17/8-chloroformylandrost-5-ene have been resolved by h.p.l.c., and the prephytoene alcohol enantiomers obtained by... 

Full details have been described of the synthesis of prephytoene alcohol (29). The synthetic route from geranylgeraniol (69) is outlined in Scheme 3. 

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. ... 

The interesting formal parallel that exists between the rearrangements of the chrysanthemyl cation and the conversion of presqualene alcohol into squalene (and now of prephytoene alcohol into phytoene) has been further explored. Solvolyses ° of the cyclopropyl (65) and cyclobutyl (63) esters both afford head-to-head coupled Cio chains analogous to squalene. A versatile new method provides access to 9-substituted p-menthanes. This starts with natural limonene and proceeds via the anion (135) which retains chirality and leads to chiral products (see below). Skeletal rearrangements in the bicycloheptane series, an historic field in the study of organic reaction mechanisms, has received a fresh impetus from the extended work of Kirmse and his colleagues, - which is of preparative and mechanistic significance. 

Prephytoene alcohol pyrophosphate (22b) has been synthesized and identified as an intermediate in the biosynthesis of phytoene (24b). The reaction sequence shown (Scheme 2 b series) assumes that the absolute stereochemistry is the same as for presqualene alcohol. 

GGPP geranylgeranyl pyrophosphate IPP isopentenyl pyrophosphate PPPP prephytoene pyrophosphate. 

It is of interest that in some nonenzymatic studies, a small percentage of head-to-head condensation products are observed. Chrysanthemyl alcohol (92) is an analog of presqua-lene alcohol and prephytoene (see Chapters 23 and 26), but the fissions postulated to lead to the irregular monoterpenes have no counterparts for the higher classes (Charlwood and Chari wood, 1991a). 

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.

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