Famesyl-diphosphate

Propose a mechanism for the biosynthesis of the sesquiterpene trichodiene from famesyl diphosphate. The process involves cyclization to give an intermediate secondary carbocation, followed by several carbocation rearrangements. 

The isoprenoid side chains of quinones are biosynthesized mainly by the mevalonic acid pathway from acetyl-CoA. Another pathway to biosynthesizing isoprenoids is the so-called non-mevalonate ronte by which isopentenyldiphosphate (IPP) is formed from glyceraldehyde 3-phosphate and pyrnvate. The key molecule is the famesyl-diphosphate (FPP) that accepts other IPP molecules to form polyprenyl diphosphates. 

Lee, PC. et al., Directed evolution of Esherichia coli famesyl diphosphate synthase (IspA) reveals novel structural determinants of chain length specihcity, Metab. Eng. 7, 18, 2005. 

Cervantes-Cervantes, M. et al., Maize cDNAs expressed in endosperm encode functional famesyl diphosphate synthase with geranylgeranyl diphosphate synthase activity, Plant Physiol. 220, 2006. 

This enzyme [EC 2.5.1.29], also known as geranylgera-nyl-diphosphate synthase, catalyzes the condensation of transqrans-famesyl diphosphate and isopentenyl diphosphate to yield geranylgeranyl diphosphate and pyrophosphate. 

The peptide substrate (400 pmol dissolved in 10 mM DTT immediately prior to use), [l-3H]famesyl diphosphate (200 pmol 15 Ci mmol-1) and farnesyltransferase (-200 units enzyme, specific activity 100-300 nmol h 1 mg1 protein) were mixed in buffer [30 pL 50 mM Tris-HCI (pH 7.7)/20 mM KQ/5 mM MgCl2/2 mM DTT]. The mixture was incubated for 20min at 37°C, then 0.1% octylglucoside was added and the reaction was allowed to proceed for 60 min. The product was purified by RP-HPLC on a C-18 column using a linear gradient of 10 to 100% MeCN in 0.1% aq TFA over 30min. Typical yield of the reaction is about 90%. 

Chain elongation during polymerization of prenyl units can be terminated in one of a number of ways. The pyrophosphate group may be hydrolyzed to a monophosphate or to a free alcohol. Alternatively, two polyprenyl compounds may join "head to head" to form a symmetric dimer. The C30 terpene squalene, the precursor to cholesterol, arises in this way from two molecules of famesyl diphosphate as does phy-toene, precursor of the Qo carotenoids, from E,E,E geranylgeranyl diphosphate. The phytanyl groups of archaebacterial lipids (p. 385) arise rather directly from geranylgeranyl diphosphate by transfer of the poly-... 

Both famesyltransferases70 761 and geranylgeranyl-transferases72/77 78b have been characterized, and the three- dimensional structure of the former has been established.73 75-76 The two-domain protein contains a seven-helix crescent-shaped hairpin domain and an a,a-barrel similar to that in Fig. 2-29. A bound zinc ion in the active site may bind the -S group of the substrate protein after the famesyl diphosphate has been bound into the active site.76 79 These enzymes are thought to function by a carbocation mechanism as shown in Eq. 22-3 and with the indicated inversion of configuration.71... 

Most of the compounds shown in Figure 22-4 are derived from the C15 famesyl diphosphate. There are more than 300 known cyclic structures among these sesquiterpenes, and many sesquiterpene synthases have been characterized.91/91a Aristolochene is formed by the action of a 38-kDa cyclase that has been isolated from species of Penicillium and Aspergillus,92"4 Notice that the synthesis must involve two cyclization steps and migration of a methyl group. Three-dimensional structures are known for at least two terpene synthases,95 96 and comparison of gene sequences suggests that many others have similar structures. 

The diterpenes arise from geranylgeranyl diphosphate (GGPP), which is formed by addition of a further IPP molecule to famesyl diphosphate in the same manner as described for the lower... 

Fig. 2 Biosynthetic route to terpenes. Geranyl diphosphate (5) famesyl diphosphate (6) gera-nylgeranyl diphosphate (7) (-)-limonene (8) (-)-camphene (9) taxadiene (10) casbene (II) capsidiol (12). IPP = isopentenyl diphosphate, DMAPP = dimethylallyl diphosphate...

Fig. 3 Proposed mechanisms for the formation of (+)-5-epi-aristolochene (2) and (-)-premnaspir-odiene (3) from famesyl-diphosphate (6) by the action of TEAS and HPS, respectively...

Figure 10.7 All terpenes are derived from allylic diphosphates which are polymers of repeating isopentyl units (IPP) put together by the action of prenyltransferases. In plants, IPP can be derived from the mevalonate biosynthetic pathway (a cytoplasmic pathway) or the methyl erythritol phosphate pathway (a plastidic pathway). Monoterpenes are then derived from the CIO precursor geranyl diphosphate (GPP), sesquiterpenes from the C15 precursor famesyl diphosphate (FPP), and diterpenes from the C20 precursor geranylgeranyl diphosphate (GGPP) by the action of terpene synthases or cyclases, which divert carbon into the specific branch pathways.

Figure 10.8 Famesyl diphosphate (FPP) can be cyclized in many different ways by sesquiterpene synthases giving rise to different classes of sesquiterpenes. The eremophilane class of sesquiterpenes has received considerable attention because the genes coding for the corresponding synthase enzymes were the first to be isolated. 25...

Due to the many applications and the industrial relevance of this class of molecules, several studies to identify strategies to increase their production have been conducted. In the recent work by Verwaal et al. [164], S. cerevisiae has been engineered with the genes from the [S-carotene production pathway from the yeast Phaffia rhodozyma, which is naturally able to produce these compounds. When these genes were cloned in Candida utilis, the carotenoids yields increased remarkably [165] and this led Verwaal et al. [164] to the idea that the production of p-carotene in S. cerevisiae could be improved by conferring properties from another yeast species. Like S. cerevisiae, P. rhodozyma also produces famesyl-diphosphate (FPP see Fig. 4) which is further converted into geranylgeranyl... 

Fig. 4 Reconstruction of the carotenoids biosynthetic pathway in S. cerevisiae. Heterologous activities t-HMGl 3 hydroxy-3-methylglutaryl-coenzymeA, ctrE geranylgeranyldiphosphate synthase (from Xanthophyllomyces dendrorhous), Ctrl phytoene desaturase, ctrYB bifunctional enzyme phytoene synthase and lycopene cyclase. Endogenous yeast activities ISIS I geranylgeranyldiphosphate synthase. Metabolites IPP isopentenyl diphosphate, GPP geranyl diphosphate, FPP famesyl-diphosphate, GGPP geranylgeranyl diphosphate...

Ohnuma S, Narita K, Nakazawa T, Ishida C, Takeuchi Y, Ohto C, Nishino T (1996) A role of the amino acid residue located on the fifth position before the first aspartate-rich motif of famesyl diphosphate synthase on determination of the final product. J Biol Chem 271 30748-30754... 

Draw famesyl diphosphate in the configuration that resembles the product, then draw its allylic isomer (the mechanism for the formation of the isomer is shown in Problem 27.7). In this reaction, a cyclization, followed by loss of a proton to form the double bond, gives helminthogermacrene. 

A second molecule of IPP adds to GPP to give famesyl diphosphate, the precursor to a-cadinene. 

Now, arrange famesyl diphosphate to resemble the skeleton of oc-cadinene. The first step in the reaction sequence is formation of the allylic isomer of FPP the mechanism was shown in Problem 27.7. 

Famesyl diphosphate (from the previous problem) dimerizes to form squalene. 

Draw famesyl diphosphate in the correct orientation in order to make this problem much easier. Internal displacement of -OPP by the electrons of one double bond is followed by attack of the electrons of the second double bond on the resulting carbocation. Loss of a proton from the carbon next to the resulting carbocation produces the double bond. 

The precursor to flexibilene is formed from the reaction of famesyl diphosphate and isopentenyl diphosphate. 

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