Presqualene pyrophosphate

Interest in presqualene pyrophosphate continues and it is claimed that the structure (88) assigned to a squalene precursor is incorrect. Presqualene pyrophosphate has been shown to contain a cyclopropyl ring (89), and both (89) and its parent alcohol have been synthesised. -Mechanisms for the conversion of (89) into squalene have been pub-lished. -... 

If the reducing agent NADPH is omitted from the cell preparation, squalene is not formed. Instead, another farnesyl pyrophosphate dimer accumulates—presqualene pyrophosphate—which has a three-membered ring and in which we can see that the two molecules of farnesyl pyrophosphate are joined in a slightly more rational way. 

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. 

Nevertheless, a number of possible pathways which bear good chemical and/or biological analogies have been proposed. For example, presqualene pyrophosphate (77) could be formed via a protonated cyclopropane intermediate (79) by direct electrophilic alkylation of the C(2,3) double bond (Scheme 3), or via a tertiary cation as suggested by... 

Since there are three possible ways to rearrange cyclopropylcarbinyl cation (86) of the type proposed in presqualene pyrophosphate conversion, and the unwanted cyclopropylcarbinyl (86) to allyl (87) rearrangement has been found to account for 99% of total reaction flux in model studiessqualene synthetase must exert strict regiochemical control in the catalytic steps to produce the enzymatic product squalene via the kinetically and thermodynamically unfavored (ca. 0.04 % of the total non-enzymatic flux) rearrangement process (86 82). A tight enzyme-substrate complex that imposes an energy barrier... 

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 ergosterol (45) biosynthesis in fungi and leishmania utilizes squalene (41) as starting material, which is obtained from long chain precursors like farnesyl pyrophosphate (39) and presqualene pyrophosphate (40). Epoxidation of squalene in the presence of squalene epoxidase furnishes squalene epoxide (42), which is succes-... 

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. ... 

The conversion of famesyl pyrophosphate to squalene is a 2-step process. First is the, l -2-3 condensation of 2 famesyl pyrophosphate molecules with the concomitant loss of a proton and inorganic pyrophosphate to form presqualene pyrophosphate [67,68]. 

The resulting cyclopropylcarbinyl pyrophosphate is reduced to squalene by NADPH. This is the head-to-head condensation of terpene biosynthesis. The absolute stereochemistry of presqualene pyrophosphate has been determined [69]. 

The enzymes necessary for the conversion of famesyl pyrophosphate to squalene are called squalene synthetase . The enzymes necessary for the two reactions have not been resolved nor has either been purified to a significant extent, and it is not yet certain if the two reactions are catalyzed by two discrete entities. Squalene synthetase has an absolute requirement for a divalent cation, Mg " and Mn " being the best. A reduced pyridine nucleotide (NADH or NADPH) is required for the reduction of presqualene pyrophosphate to squalene. Yeast microsomes with Mn " and no reduced pyridine nucleotide will form dehydrosqualene instead [70]. The conversion of famesyl pyrophosphate to presqualene pyrophosphate is enhanced several-fold by the reduced pyridine nucleotide [71]. Also, some organic solvents as well as detergents increase this activity. 

The kinetics of the overall reaction are controversial as might be expected since the substrate is water soluble while the intermediate and the product are not. What can be said is that under the right conditions there is stoichiometry between presqualene pyrophosphate, squalene and farnesyl pyrophosphate. In addition, since high concentrations of farnesyl pyrophosphate transitorily inhibit the second reaction, and NADPH stimulates the first, it can be concluded that the enzyme(s) has separate but closely related sites for the two reactions. These sites could be on one or two peptide chains [71],... 

Fig. 14. The condensation between 2 molecules of farnesyl pyrophosphate (A, acceptor B, donor). The product is presqualene pyrophosphate (C).

Fig. 15. A cyclopropylammonium analog of presqualene. As the pyrophosphate, it may resemble a transition state between presqualene pyrophosphate and squalene.

Fig. 16. Two possible pathways for rearrangements leading to the formation of squalene from presqualene pyrophosphate.

The head-to-head union of two farnesyl pyrophosphate molecules to produce squalene is promoted by squalene synthase (33). The enzyme utilizes NADPH as a cofactor. If the cofactor is omitted, another species, presqualene pyrophosphate, accumulates (34). On addition of NADPH, this is converted to squalene. The structure of presqualene pyrophosphate was elucidated by Epstein and Rilling, who found that the material contains a highly substituted cyclopropane ring as a central feature (35). Poulter and associates (36), as well as van Tamelen and... 

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