B. Biosynthetic Origins of Sterols

Scheme 1



Synthesis of mevalonic acid from acetyl CoA.



degradation, which are in turn regulated by the amount of cholesterol in the cell.

Cholesterol content is influenced by the rate of biosynthesis, dietary uptake, and a

lipoprotein system that traffics in the intercellular movement of cholesterol. During

growth, cholesterol is mainly incorporated into the cell membrane. However, in homeostasis cholesterol is mainly converted to bile acids and is transported to other

tissues via low density lipoprotein (LDL). High density lipoprotein (HDL) also serves

as a cholesterol carrier, which carries cholesterol from peripheral tissues to the liver.

The major metabolic route of cholesterol is its conversion to bile acids and neutral

sterols, which are excreted from the liver via the bile. Kandutsch and Chen and

others have shown that oxysterols regulate the biosynthesis of HMG-CoA reductase

as well as its digression, which controls cholesterol biosynthesis [37]; the regulation

of HMG-CoA reductase by oxysterols is discussed in more detail in a later section.

A number of substrate analogs have been tested for their inhibition of HMG-CoA

reductase. Some of them (e.g., compactin and melinolin) were found to be very

effective in treating hypocholesterol diseases [38,39].

The coupling of six molecules of mevalonic acid to produce squalene proceeds

through a series of phosphorylated compounds. Mevalonate is first phosphorylated

by mevalonic kinase to form a 5-phosphomevalonate, which serves as the substrate

for the second phosphorylation to form 5-pyrophosphomevalonate (Scheme 2). There

is then a concerted decarboxylation and loss of a tertiary hydroxy group from 5pyrophosphomevalonate to form 3-isopentyl pyrophosphate, and in each step one

molecule of ATP must be consumed. 3-Isopentyl pyrophosphate is regarded as the

basic biological ‘‘isoprene unit’’ from which all isoprenoid compounds are elaborated. Squalene is synthesized from isopentyl pyrophosphate by sequence coupling

reactions. This stage in the cholesterol biosynthesis starts with the isomerization of

isopentyl pyrophosphate to dimethylallyl pyrophosphate. The coupling reaction

shown in Scheme 2 is catalyzed by a soluble sulfydryl enzyme, isopentyl pyrophosphate–dimethylallyl pyrophosphate isomerase. The coupling of these two isomeric



Copyright 2002 by Marcel Dekker, Inc. All Rights Reserved.



Scheme 2 Synthesis of isopentenyl pyrophosphate, the biological ‘‘isoprene unit,’’ and

dimethylallyl pyrophosphate.



C5 units yields geranyl pyrophosphate, which is catalyzed by geranyl pyrophosphate

synthetase (Scheme 3). This reaction proceeds by the head-to-tail joining of isopentyl

pyrophosphate to dimethylallyl pyrophosphate. A new carbon–carbon bond is formed

between the C-1 of dimethylallyl pyrophosphate and C-4 of isopentyl pyrophosphate.

Consequently, geranyl pyrophosphate can couple in a similar manner with a second

molecule of isopentyl pyrophosphate to produce farnesyl pyrosphate (C15 structure).

The last step in the synthesis of squalene is a reductive condensation of two molecules of farnesyl pyrophosphate (Scheme 4). This step is actually a two-step sequence, catalyzed by squalene synthetase. In the first reaction, presqualene pyrophosphate is produced by a tail-to-tail coupling of two farnesyl pyrophosphate

molecules. In the following conversion of presqualene pyrophosphate to squalene,

the cyclopropane ring of presqualene pyrophosphate is opened with a loss of the

pyrophosphate moiety. A molecule of NADPH is required in the second conversion.

The third stage of cholesterol biosynthesis is the cyclization of squalene to

lanosterol (Scheme 5). Squalene cyclization proceeds in two steps requiring, molecular oxygen, NADPH, squalene epoxidase, and 2,3-oxidosqualene–sterol cyclase.

The first step is the epoxidation of squalene to form 2,3-oxidosqualene–sterol cyclase. The 2,3-oxidosqualene is oriented as a chair–boat–chair–boat conformation

in the enzyme active center. The acid-catalyzed epoxide ring opening initiates the

cyclization to produce a tetracyclic protosterol cation. This is followed by a series

of concerted 1,2-trans migrations of hydrogen and methyl groups to produce

lanosterol.

The last stage of cholesterol biosynthesis is the metabolism of lanosterol to

cholesterol. Scheme 6 gives the general biosynthetic pathway from lanosterol to



Copyright 2002 by Marcel Dekker, Inc. All Rights Reserved.



Scheme 3



Synthesis of farnesyl pyrophosphate from the biological ‘‘isoprenyl unit.’’



cholesterol. The C-14 methyl group is first oxidized to an aldehyde, and removed as

formic acid. The oxidation of the C-4␣ methyl group leads to an intermediate, 3oxo-4␣ -carboxylic acid, which undergoes a decarboxylation to form 3-oxo-4␤ -methylsterol. This compound is then reduced by an NADPH-dependent microsomal 3oxosteroid reductase to produce 3␤ -hydroxy-4␣ -methyl sterol, which undergoes a

similar series of reactions to produce a 4,4-dimethylsterol. In animal tissues, C-14

demethylation and the subsequent double-bond modification are independent of the

reduction of the ⌬24 double bond. Desmosterol (cholesta-5,24-dien-3␤ -ol) is found

in animal tissues and can serve as a cholesterol precursor. The double-bond isomerization of 8 to 5 involves the pathway ⌬8 → ⌬7 → ⌬5,7 → ⌬5.

2.



Biosynthesis of Plant Sterols



In animals, 2,3-oxidosqualene is first converted to lanosterol through a concerted

cyclization reaction. This reaction also occurs in yeast. However, in higher plants

and algae the first cyclic product is cycloartenol (Scheme 7). The cyclization intermediate, tetracyclic protosterol cation, undergoes a different series of concerted 1,2trans migrations of hydrogen and methyl groups. Instead of the 8,9 double bond, a

stabilized C-9 cation intermediate is formed. Following a trans elimination of enzyme-X Ϫ and Hϩ from C-19, with the concomitant formation of the 9,19-cyclopropane ring, cycloartenol is formed. A nearby ␣-face nucleophile from the enzyme is

necessary to stabilize the C-9 cation and allow the final step to be a trans elimination



Copyright 2002 by Marcel Dekker, Inc. All Rights Reserved.



Scheme 4



Synthesis of squalene from the coupling of two molecules of farnesyl

pyrophosphate.



according to the isoprene rule. The biosynthesis pathway from acetyl CoA to 2,3oxisqualene in plants is the same as that in animals (see detailed discussion of the

biosynthesis of cholesterol, Sec. II.B.1).

The conversion of cycloartenol to other plant sterols can be generally divided

into three steps, which are the alkylation of the side chain at C-24, demethylation



Scheme 5



Cyclization of squalene.



Copyright 2002 by Marcel Dekker, Inc. All Rights Reserved.



Scheme 6



Biosynthesis of cholesterol from lanosterol.



of the C-4 and C-14 methyl groups, and double-bond manipulation. Alkylation in

the formation of plant sterols involves methylation at C-24 with S-adenosylmethionine to produce C28 sterols. The further methylation of a C-24 methylene substrate

yields C-24 ethyl sterols. The details of the mechanism of demethylation and doublebond manipulation in plants are not clear, but it is highly likely to be very similar

to that in animals. In plants, C-4 methyl groups are removed before the methyl group

at C-14, whereas in animals it is the other way around. Sterols found in plants

are very diversified. The structural features of major plant sterols are depicted in

Figure 2.

C.



Regulation of Sterol Biosynthesis in Animals



Sterol biosynthesis in mammalian systems has been intensely studied for several

decades. Interest in the cholesterol biosynthesis pathway increased following clinical

observations that the incidence of cardiovascular disease is greater in individuals

with levels of serum cholesterol higher than average. More recently, the results of

numerous clinical studies have indicated that lowering serum cholesterol levels may



Copyright 2002 by Marcel Dekker, Inc. All Rights Reserved.



Scheme 7



Figure 2



Cyclization of squalene to cycloartenol.



Examples of plant sterols.



Copyright 2002 by Marcel Dekker, Inc. All Rights Reserved.