Biosynthesis of Cholesterol, Steroids, and Isoprenoids:- Cholesterol Is Made from Acetyl-CoA in Four Stages
Cholesterol, like long-chain fatty acids, is made from acetyl-CoA, but the assembly plan is quite different. In early experiments, animals were fed acetate labeled with 14C in either the methyl carbon or the carboxyl car bon. The pattern of labeling in the cholesterol isolated from the two groups of animals (Fig. 1) provided the blueprint for working out the enzymatic steps in cholesterol biosynthesis.
Synthesis takes place in four stages, as shown in Figure 1: 1 condensation of three acetate units to form a six-carbon intermediate, mevalonate; 2 con version of mevalonate to activated isoprene units; 3 polymerization of six 5-carbon isoprene units to form the 30-carbon linear squalene; and 4 cyclization of squalene to form the four rings of the steroid nucleus, with a further series of changes (oxidations, removal or migration of methyl groups) to produce cholesterol.
FIGURE 1 Origin of the carbon atoms of cholesterol. This can be deduced from tracer experiments with acetate labeled in the methyl carbon (black) or the carboxyl carbon (red). The individual rings in the fused-ring system are designated A through D.
FIGURE 2 Summary of cholesterol biosynthesis. The four stages are discussed in the text. Isoprene units in squalene are set off by red dashed lines.
Stage 1 Synthesis of Mevalonate from Acetate The first stage in cholesterol biosynthesis leads to the intermediate mevalonate (Fig. 3). Two molecules of acetyl-CoA condense to form acetoacetyl-CoA, which condenses with a third molecule of acetyl-CoA to yield the six-carbon compound β-hydroxy- β -methyl glutaryl-CoA (HMG-CoA). These first two reactions are catalyzed by thiolase and HMG-CoA synthase, re spectively. The cytosolic HMG-CoA synthase in this pathway is distinct from the mitochondrial isozyme that catalyzes HMG-CoA synthesis in ketone body formation (see Fig. 17–18).
The third reaction is the committed and rate-limiting step: reduction of HMG-CoA to mevalonate, for which each of two molecules of NADPH donates two electrons. HMG-CoA reductase, an integral membrane protein of the smooth ER, is the major point of regulation on the pathway to cholesterol, as we shall see.
FIGURE 3 Formation of mevalonate from acetyl-CoA. The ori gin of C-1 and C-2 of mevalonate from acetyl-CoA is shown in pink.
Stage 2 Conversion of Mevalonate to Two Activated Isoprenes In the next stage of cholesterol synthesis, three phosphate groups are transferred from three ATP molecules to mevalonate (Fig. 4). The phosphate attached to the C-3 hydroxyl group of mevalonate in the intermediate 3-phospho-5-pyrophosphomevalonate is a good
FIGURE 4 Conversion of mevalonate to activated isoprene units. Six of these activated units combine to form squalene (see Fig. 5). The leaving groups of 3-phospho-5-pyrophosphomevalonate are shaded pink. The bracketed intermediate is hypothetical.
leaving group; in the next step, both this phosphate and the nearby carboxyl group leave, producing a double bond in the five-carbon product, Δ3-isopentenyl pyrophosphate. This is the first of the two activated isoprenes central to cholesterol formation. Isomerization of Δ3-isopentenyl pyrophosphate yields the second activated isoprene, dimethylallyl pyrophosphate. Synthesis of isopentenyl pyrophosphate in the cytoplasm of plant cells follows the pathway described here. However, plant chloroplasts and many bacteria use a mevalonate independent pathway. This alternative pathway does not occur in animals, so it is an attractive target for the development of new antibiotics.
Stage 3 Condensation of Six Activated Isoprene Units to Form Squalene Isopentenyl pyrophosphate and dimethylallyl pyrophosphate now undergo a head-to-tail condensation, in which one pyrophosphate group is displaced and a 10-carbon chain, geranyl pyrophosphate, is formed (Fig. 5). (The “head” is the end to which pyrophosphate is joined.) Geranyl pyrophosphate undergoes another head-to-tail condensation with isopentenyl pyro phosphate, yielding the 15-carbon intermediate farnesyl pyrophosphate. Finally, two molecules of farnesyl pyrophosphate join head-to-head, with the elimination of both pyrophosphate groups, to form squalene. The common names of these intermediates derive from the sources from which they were first isolated. Geraniol, a component of rose oil, has the aroma of geraniums, and farnesol is an aromatic compound found in the flowers of the Farnese acacia tree. Many natural scents of plant origin are synthesized from isoprene units. Squalene, first isolated from the liver of sharks (genus Squalus), has 30 carbons, 24 in the main chain and 6 in the form of methyl group branches.
Stage 4 Conversion of Squalene to the Four-Ring Steroid Nucleus When the squalene molecule is represented as in Figure 6, the relationship of its linear structure to the cyclic structure of the sterols becomes apparent. All
FIGURE 6 Ring closure converts linear squalene to the condensed steroid nucleus. The first step in this sequence is catalyzed by a mixed-function oxidase (a monooxygenase), for which the co substrate is NADPH. The product is an epoxide, which in the next step is cyclized to the steroid nucleus. The final product of these reactions in animal cells is cholesterol; in other organisms, slightly different sterols are produced, as shown.
sterols have the four fused rings that form the steroid nucleus, and all are alcohols, with a hydroxyl group at C-3—thus the name “sterol.” The action of squalene monooxygenase adds one oxygen atom from O2 to the end of the squalene chain, forming an epoxide. This enzyme is another mixed-function oxidase ; NADPH reduces the other oxygen atom of O2 to H2O. The double bonds of the product, squalene 2,3-epoxide, are positioned so that a remarkable concerted reaction can convert the linear squalene epoxide to a cyclic structure. In animal cells, this cyclization results in the formation of lanosterol, which contains the four rings characteristic of the steroid nucleus. Lanosterol is finally converted to cholesterol in a series of about 20 reactions that include the migration of some methyl groups and the removal of others. Elucidation of this extraordinary biosynthetic pathway, one of the most complex known, was accomplished by Konrad Bloch, Feodor Lynen, John Cornforth, and George Popják in the late 1950s. Cholesterol is the sterol characteristic of animal cells; plants, fungi, and protists make other, closely related sterols instead. They use the same synthetic path way as far as squalene 2,3-epoxide, at which point the pathways diverge slightly, yielding other sterols, such as stigmasterol in many plants and ergosterol in fungi (Fig. 6).
