Lanosterol pathway, sterols

Triterpenoid saponins are synthesized via the isoprenoid pathway.4 The first committed step in triterpenoid saponin biosynthesis involves the cyclization of 2,3-oxidosqualene to one of a number of different potential products (Fig. 5.1).4,8 Most plant triterpenoid saponins are derived from oleanane or dammarane skeletons although lupanes are also common 4 This cyclization event forms a branchpoint with the sterol biosynthetic pathway in which 2,3-oxidosqualene is cyclized to cycloartenol in plants, or to lanosterol in animals and fungi. 

Analysis of the details of the pathway was helped by the discovery by Nancy Bucher (1953) that cholesterol synthesis took place in cell-free post-mitochondrial supernatants. ATP, Mg2+ and NAD+ were required. Tchen and Bloch extended these findings to show that squalene could be formed anaerobically but the conversion of squalene to cholesterol was oxygen dependent, the oxygen of the intermediate lanosterol being derived from 8C>2 not H2180. It therefore became possible to focus either on the conversion of acetate to squalene or on the latter s cyclization to the sterol. 

Wood SG, Gottheb D (1978) Evidence from mycelial studies for differences in the sterol biosynthetic pathway of Rhizoctonia solani and Phytophthora cinnamomi. Biochem J 170 343 Nes WD et al (1986) A comparison of cycloartenol and lanosterol biosynthesis and metabolism by the Oomycetes. Expeiientia 42 556... 

The synthesis of all isoprenoids starts with acetyl-CoA, which in a series of six different enzyme reactions is converted into isopentenyl-diphosphate (-PP), the basic C-5 isoprene unit that is used for the synthesis of all subsequent isoprenoids (Fig. 5.1.1). At the level of farnesyl-PP the pathway divides into several branches that are involved in the production of the various isoprenoid end products. One of the major branches involves the cholesterol biosynthetic part of the pathway, of which squalene is the first committed intermediate in the production of sterols. Following cycliza-tion of squalene, lanosterol is produced. To eventually produce cholesterol from la-... 

The rearrangement of this initially created C-20 carbocation to lanosterol (Fig. 22-6, step c) is also a remarkable reaction that requires the shift of a hydride ion and of two methyl groups, as indicated by the arrows in the figure. In addition, a hydrogen at C-9 (sterol numbering) is lost as a proton. Lanosterol is named for its occurrence in lanolin, the waxy fat in wool. Although the principal component of lanolin is cholesterol, lanosterol is its precursor both in sheep and in all other animals. Cholesterol is in turn the precursor to other animal sterols. The cholesterol biosynthetic pathway also provides cells with a variety of important signaling molecules.1603... 

The last sequence of reactions in the biosynthesis of choles-terol involves approximately 20 enzymatic steps, starting with lanosterol. In mammals the major route involves a series of double-bond reductions and demethylations (fig. 20.10). The sequence of reactions involves reduction of the A24 double bond, the oxidation and removal of the 14a methyl group followed by the oxidation and removal of the two methyl groups at position 4 in the sterol. The final reaction is a reduction of the A7 double bond in 7-dehydro-cholesterol. An alternative pathway from lanosterol to cholesterol also exists. The enzymes involved in the transformation of lanosterol to cholesterol are all located on the endoplasmic reticulum. 

A number of cytochrome P450 enzymes are involved in the conversion of acetate to sterols and bile acids (Figure 9.6). The participation of P450 enzymes in pathways of cholesterol biosynthesis and elimination demonstrate their important role in cholesterol homeostasis. Lanosterol 14a-desmethylase, encoded by CYP51A1, is a pivotal P450 involved in cholesterol biosynthesis. The synthesis of bile acids from... 

Cycloartenol (94) rather than lanosterol is thought to be the crucial triterpenoid intermediate in the biosynthesis of plant sterols, although in a chlorophyll-containing phylum there appears to be no direct correlation between ability to photosynthesize and the operation of the cycloartenol pathway.163 The cleavage of the cyclopropane ring of (94) between C-9 and C-19 should be accompanied by the incorporation of a proton at C-19, possibly from the medium. That this is the case in pea was shown164 by the incorporation of deuterium specifically at C-19 of cycloartenol obtained from... 

Figure 1.7 illustrates the synthesis of sterols in yeasts. Basically, sterols are synthesised by the mevalonate pathway. The key stage in this pathway is, without any doubt, the reaction catalysed by squalene monooxygenase. This reaction, which uses oxygen as substrate, transforms squalene into squalene 2,3, epoxide. Later, squalene epoxide lanosterol cyclase catalyses the synthesis of the first sterol of the pathway. 

Grausem, B., Ghaubet, N., Gigot, G., Loper, J. and Benveniste, P. (1995) Functional expression of Daccharomyces cerevisiae CYP51A1 encoding lanosterol-14-demethylase in tobacco results in bypass of endogenous sterol biosynthetic pathway and resistance to an obtusifoliol-14-demethylase herbicide. Plant J., 7, 761-70. 

Evidence has been accumulated that the biogenesis of plant steroids proceeds by the same scheme as in animals this sequence was proved up to squalene and recently squalene has been shown to be a precursor of, 8-sitosterol evidence was also adduced that this conversion includes lanosterol as an intermediate (233). Goutarel assumes that the sequence squalene, lanosterol, zymosterol, and desmosterol may be implied also in the biogenetic pathway of Apocynaceae alkaloids (205). It may be assumed that biogenetic intermediates from sterol or triterpene precursors of Apocynaceae or Buxaceae alkaloids, respectively, are 3-and/or 20-ketones which may give rise to mono- and diamines. 

In our laboratory, the major sterol biosynthesised in untreated extracts was the triene (VIII) (1J), rather than ergosterol Itself which is, of course, the end product of the pathway in intact cells. It should be noted that VIII arises directly from 14-demethylation of lanosterol. In the presence of 0.1 yM prochloraz (or even 0.01 pM prochloraz in some experiments) the concentration of VIII was significantly reduced, while the level of lanosterol Increased (4) indicating clearly that prochloraz inhibited 14-demethylation. At higher fungicide concentrations both the triene and ergosterol were totally absent and only lanosterol was present. 

Since ergosterol is used in the formation of the leishmanial cell membrane, inhibition of ergosterol biosynthesis has been considered as a useful target for chemotherapeutic attack. Allylamines (eg. terbinafine) and imidazole antifungals (eg. ketoconazole) have been found to interfere with different steps in the biosynthetic pathway of C28 sterols in leishmania and fungi. Allylamines inhibit the microsomal squalene 2,3-epoxidase and, therefore, inhibit the synthesis of squalene epoxide, the precursor of lanosterol. Imidazoles, on other hand, inhibit cytochrome P-450 dependent C-14 demethylation of lanosterol leading to decreased or no synthesis of ergosterol [30]. 

Relatively little is known about plant sterols. (Most of the research effort in steroid metabolism has been expended in the investigation of steroid-related human diseases.) It appears, however, that the initial phase of plant sterol synthesis is very similar to that of cholesterol synthesis with the following exception. In plants and algae the cyclization of squalene-2,3-epoxide leads to the synthesis of cycloartenol (Figure 12.30) instead of lanosterol. Many subsequent reactions in plant sterol pathways involve SAM-mediated methylation reactions. There appear to be two separate isoprenoid biosynthetic pathways in plant cells the ER/cyto-plasm pathway and a separate chloroplast pathway. The roles of these pathways in plant isoprenoid metabolism are still unclear. 

The enzymes responsible for the conversion of lanosterol to cholesterol, as were those for the conversion of farnesyl pyrophosphate to squalene and lanosterol, are all integral membrane-bound proteins of the endoplasmic reticulum. Many have resisted solubiUzation, some have been partially purified, and several have been obtained as pure proteins. As a consequence, much of the enzymological and mechanistic studies have been done on impure systems and one would anticipate a more detailed and improved understanding of these events as more highly purified enzymes become available. Many approaches have been taken to establish the biosynthetic route that sterols follow to cholesterol. Some examples are synthesis of potential intermediates, the use of inhibitors, both of sterol transformations and of the electron transfer systems, and by isotope dilution experiments. There is good evidence that the enzymes involved in these transformations do not have strict substrate specificity. As a result, many compounds that have been found to be converted to intermediates or to cholesterol may not be true intermediates. In addition, there is structural similarity between many of the intermediates so that alternate pathways and metabolites are possible. For example, it has been shown that side-chain saturation can be either the first or the last reaction in the sequence. Fig. 21 shows a most probable series of intermediates for this biosynthetic pathway. 

Squalene is converted into the first sterol, lanosterol, by the action of squalene epoxidase and oxidosqualene lanosterol cyclase. The catalytic mechanism for the cyclase s four cyclization reactions was revealed when the crystal stmcture of the human enzyme was obtained (R. Thoma, 2004). Oxidosqualene lanosterol cyclase is considered an attractive target for developing inhibitors of the cholesterol biosynthetic pathway because its inhibition leads to the production of 24,25-epoxycholesterol (M.W. Huff, 2005). This oxysterol is a potent ligand activator of the liver X receptor (LXR) and leads to expression of several genes that promote cellular cholesterol efflux, such as ABCAl, ABCG5, and ABCG8 (Section 4.1). Thus, inhibitors of oxidosqualene lanosterol cyclase could be therapeutically advantageous because they would reduce cholesterol synthesis and promote cholesterol efflux (M.W. Huff, 2005). 

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