1

2. Biosynthesis of Natural Products - Terpene Biosynthesis

2.1 Introduction
Terpenes are a large and varied class of natural products, produced primarily by a wide variety of plants,
insects, microoroganisms and animals. They are the major components of resin, and of turpentine produced
from resin. The name "terpene" is derived from the word "turpentine". Terpenes are major biosynthetic
building blocks within nearly every living creature. Steroids, for example, are derivatives of the triterpene
squalene. When terpenes are modified, such as by oxidation or rearrangement of the carbon skeleton, the
resulting compounds are generally referred to as terpenoids. Some authors will use the term terpene to
include all terpenoids. Terpenoids are also known as Isoprenoids.

Terpenes and terpenoids are the primary constituents of the essential oils of many types of plants and
flowers. Essential oils are used widely as natural flavor additives for food, as fragrances in perfumery, and
in traditional and alternative medicines such as aromatherapy. Synthetic variations and derivatives of natural
terpenes and terpenoids also greatly expand the variety of aromas used in perfumery and flavors used in
food additives. Recent estimates suggest that over 30'000 different terpenes have been characterized from
natural sources.

Early on it was recognized that the majority of terpenoid natural products contain a multiple of 5C-atoms.
Hemiterpenes consist of a single isoprene unit, whereas the monoterpenes include e.g.:
Monoterpenes

CH2OH CHO

CH2OH
OH

Myrcens Limonens Nerol Geraniol Citral Menthol

O
CHO O

Camphor
!-Pinene
Citronellal

Terpenes with 15 C-atoms are known as sesquiterpenes :

Sesquiterpenes

CH2OH

Farnesol Bisabolene Cadinene Selinene Vetivone

OH
HO
COOH COOMe

OH Abscisic acid
O (Phytohormone) O
Patchoulol
(Perfume) O O
Pentalenolactone
(Antibiotic)
The terpenes containing, or originating from precursors, containing 20 C-atoms are known as diterpenes,
those with 30 C-atoms as triterpenes and those with 40 C-atoms as tetraterpenes :
 2

Diterpenes
Ph AcO O OH
O
CH2OH NH O

Phytol Ph O O
OH H
CH2OH OAc
Vitamin A OH OBz
(Retinol)
Taxol (anti-cancer)

OH Giberellic acid
(Phytohormone)
O
O
Casbene
HO
(Phytoalexin) COOH

Squalene
Triterpene

COOH O
HO CH2O
H
O
OH
H H H

H H H H H H
HO HO OH O
H
Cholesterol Cortisone
(Membrane component) Cholic acid
(Hormone)
Testosterone OH OH O
(Hormone) stradiol Progesterone
(Hormone) (Hormone)
H H
H
H H H H
H H
O HO
O

In contrast to other classes of terpenes that vary greatly in structure and molecular size, the steroids
constitute a family of terpenes with a common structural feature, namely, the steroid ring system:

Tetraterpene

-Carotene
(Pigment, Provitamin A)
 3

Mixed origin Me
O Plastoquinone Me
(Electron transport) Chlorophyll-a N N
(Photosynthesis) Mg
N N
O
18 Me Me

OH O
O COOMe
O
Tetrahydrocannabinol
O C5H11 (Cannabis sativa)

Polymer Rubber
(Heva brasilensis)
OH
500-5000

Ruzicka (ETH-ZH) recognized already in the 1920's that most terpenes appear to be constructed from a
multiple of linked isoprene units. This is called the isoprene rule.

The isoprene rule (cf. Birch, Polyketide Hypothesis) was of great value also in the structure determination
of new terpenoids isolated from Nature. However, isoprene itself is not the building block used by Nature to
construct terpenes.

O OH
Camphor

OH
CH2OH Grandisol

Menthol
Cadinene Vitamin A

2.2 The Mevalonate Pathway

It was only much later (ca. 1955) shown that the biosynthesis of terpenes does indeed occur starting from
isoprene-like C5 building blocks. Labelling experiments, using 14C-labelled acetic acid, showed early on
that the steroid skeleton is constructed from this building block, but not simply through regular head-to-tail
coupling reactions:
Me Me
Me
Me
Me COOH Me
H

H H
HO
 4
A breakthrough came around 1955 with the discovery of mevalonic acid (MVA), which was isolated from
concentrated yeast extracts at the end of the beer brewing process. It was also shown that 14C-labelled forms
of MVA are efficiently and specifically incorporated into cholesterol. Another important discovery was the
isolation and structure determination of squalene from sharks (Squalus), which was also shown to be an
efficient biosynthetic intermediate in steroid biosynthesis :

Me Me
Me
Me
Me COOH Me
H

H H
HO
Me
Me H
Me Me
Me
Me OH Me
H Me
Me Me
Me
HOOC OH Me Me
HO
Me Me Me
Me

In the mean time, all the steps from acetyl-CoA to cholesterol have been established and most of the
enzymes involved in the pathway have been isolated and studied. The pathway from acetyl-CoA to MVA,
and on to the various classes of terpenes has now been discovered in almost all living organisms, and is
known as the mevalonate pathway :

O O O O O
+ +
Me SCoA Me SCoA Me SCoA Me SCoA

Me OH Me OH
Reduction 2x with NADPH
+ CoASH

O OH O OH O SCoA
OH
(R)-Mevalonic acid O
-P- = P O
O-
3 ATP Me C5-building blocks Me
CO2
+ O-P-P
O-P-P Me

3 ADP Isopentenyl pyrophosphate Dimethylallyl pyrophosphate

(IPP) (DMAPP)

The enzyme 3-hydroxy-3-methylglutaryl-CoA synthase catalyzes an Aldol-type reaction that is unusal from
a regiochemical viewpoint:
Me OH
O O O
+
Me SCoA Me SCoA + CoASH
O OH O SCoA
 5
Mechanism:
O
O
CoASH
SCoA S
SH

A H
O O
O O
SCoA
S S
H

B
O HO Me O
H2O + HMGS
O HO Me O
HO SCoA
S SCoA

Through crystallographic studies, also with substrates bound at the active site, a good model for the reaction
mechanism has been established. The structures have also shown which residues at the active site are most
likely involved in catalysis (Vgl PNAS 2004, 101, 16442):

A. Acetoacetyl-CoA and Acetyl-Cys, and

B. HMG-CoA in the active site

In the next step of the mevalonate pathway, the CoAS-thioester group is reduced in a reaction requiring two
equivalents of NADPH. The reaction proceeds in two steps (thioester aldehyde alcohol). Many
inhibitors of this enzymic reaction have been discovered, and several of these (called statins) are now
important pharmaceutical products. The statins (or HMG-CoA reductase inhibitors) form a class of
hypolipidemic drugs used to lower cholesterol levels in people with, or at risk of, cardiovascular disease.
 6
They lower cholesterol by inhibiting the enzyme HMG-CoA reductase (HMGR), which is the rate-limiting
enzyme of the mevalonate pathway of cholesterol synthesis.

In the 1970's the Japanese microbiologist Akira Endo first discovered natural products with a powerful
inhibitory effect on HMGR in a fermentation broth of Penicillium citrinum, during the search for
antimicrobial agents. The first product was named compactin (ML236B or mevastatin). Animal trials
showed very good inhibitory effects, however, in a long term toxicity study in dogs toxic effects were
observed at higher doses. In 1978, Alfred Alberts and colleagues at Merck Research Laboratories
discovered a new natural product in a fermentation broth of Aspergillus terreus, their product showed good
HMGR inhibition and they named the product mevinolin, which later became known as lovastatin.

$! !
$! !
$!
! ! &!!
! !
!$
! $
"# $ !
"# "# $ %&'(
"#
$! !
"# $! ! !
Compactin (IC50 = 23 nM)
! !$
Mevinolin !$
(Lovastatin) *
$! !
*
"# ! ) ! )
!
%&'(
HMG-CoA (Km = 4 M) Cerivastatin (IC50 = 10 nM)
Fluvastatin (IC50 = 28 nM)

The essential structural components of all statins are a dihydroxyheptanoic acid unit and a ring system with
different substituents. The statin pharmacophore is a modified hydroxyglutaric acid component, which is
structurally similar to the endogenous substrate HMG-CoA and the mevaldyl-CoA intermediate in the
enzymic reaction. The statin pharmacophore binds to the same active site as the substrate HMG-CoA and
inhibits the HMGR enzyme. It has also been shown that the HMGR is stereoselective and as a result all
statins need to have the 3R,5R absolute configuration.

Subsequent steps lead to the important C5 building blocks IPP and DMAPP. IPP is isomerized to DMAPP
by the enzyme isopentenyl pyrophosphate isomerase:

During the past 10 years a very important discovery was made, namely, that in some organisms an
alternative pathway exists to DMAPP and IPP. This alternative pathway is found in some microorganisms
 7
as well as the plastids of plants and algae, and is called the MEP (2-methyl-D-erythritol-4-phosphate)-
pathway (or more simply the non-mevalonate pathway), which is initiated from C5-sugars. The
mechanisms of some of the steps in this pathway have not yet been elucidated:

Me
CO2 Me
O
COOH O HO Me CTP PPi HO Me O
NADPH O P O CMP
HO O PO3 2-
CHO TPP HO OH O-
OH HO OH
OH
CH2 O-PO32- ATP
CH2 O-PO3
Deoxyxylulose-
5-Phosphate ADP
PO2 O
Me Me O 2-O PO Me
3 O
PO2
O P 2O63- O O P O CMP
HO O-
H2O H+- HO OH
CMP
HO OH
2e
H+ 2e-
Me

H2O Me O P 2O63- DMAPP

Me

O P 2O63- IPP

After the formation of IPP and DMAPP, there exists in all organisms a central route to the universal
building blocks needed for mono-, sesqui-, di-, tri and tetra-terpene biosynthesis:
C10-Building block
Me Me
Me Me Monoterpenes
+
Me O-P-P O-P-P Me O-P-P
IPP Geranyl pyrophosphate
DMAPP (GPP)
C15-Building block
Me Me Me Me Sesquiterpenes
Me
O-P-P + O-P-P Me O-P-P
R
GPP IPP Farnesyl pyrophosphate
(FPP)
C20-Building block
Me Me Me Me Me
Me Diterpenes
+ O-P-P
R O-P-P Me O-P-P
IPP Geranylgeranyl pyrophosphate
FPP (GGPP)

Me Me Me C30-Building block
Me + P-P-O R
Me
O-P-P Me
R Me
Me Me Me
FPP FPP Squalene
Triterpenes
Me + P-P-O R Steroids
R O-P-P C40-Building block
Me Tetraterpenes
GGPP GGPP

The mechanism and stereochemical course of all these steps was investigated by J. W. Cornforth, who
received the Nobel Prize in Chemistry for his work (1975, mit V. Prelog, ETH-ZH). In recent years, direct
access to the biosynthetic genes for many of the enzymes in terpene biosynthesis has provided an enormous
impulse for structural and mechanistic studies. There is also great interest in the design and development of
specific inhibitors, as potential drugs against bacterial and parasitic infections, and in the agrochemical area.
 8
2.3 The Formation of GPP, FPP und GGPP
The steps from DMAPP and IPP to GPP, FPP and GGPP are catalyzed by so-called prenyl transferases.
These enzymes (35 - 80 kDa) require Metal2+-ions for activity. The Km values are typically <10M und kcat
values are in the range 0.03-0.3 s-1. In the active site of each enzyme, the pyrophosphate group is activated
and acts as a leaving group to generate an allylic-tertiary carbocation, stabilized in an ion pair with the
pyrophosphate group. This electrophile is then attacked by the double bond in the neighboring substrate

Me
Me

CF3 O-P-P
O-P-P
HR HS
Me

Me O-P-P

The reaction proceeds stereospecifically - only the pro-R H-atom is lost, and the new C=C double bond has
the E configuration. A trifluoromethyl analogue of the substrate GPP reacts 106-times slower, which
supports the mechanism involving formation of a carbocation, since formation of this would now be
destabilized by a strong inductive effect.

2.4. The Formation of Squalene

Here two FPP molecules are combined in a head-to-head coupling, which requires NADPH :

Me R Me H H
H H P-P-O
R
R O-P-P H H NADPH NADP + R
Me
H H Me
During assays in vitro, if NADPH is not present an intermediate can be detected, which normally does not
accumulate:

FPP
OPP H OPP
H H OPP
HS
HR Me

R Me Me R
R Me R R Me

Presqualen-pyrophosphat
H OPP H

Me Me

R R
R Me R Me R Me Me R

2.5. The Formation of Mono-, Sesqui- und Di-Terpenes (see also: Topics in Current Chemistry, Vol 209
Biosynthesis: Aromatic Polyketides, Isoprenoids, Alkaloids. Springer Verlag, 2000)
The terpene cyclases form a large family of enzymes that use GPP, FPP or GGPP as substrate and catalyze
the formation of a mono-, sesqui- or di-terpenoid products. The cyclases are similar in mode of action to the
 9
prenyl transferases, except now mostly intramolecular cyclization reactions are catalyzed. This family of
enzymes catalyze a huge variety of different transformations, which may include steps where H-atom
migrations, Wagner-Meerwein rearrangements, and related reactions occur. The chemistry is dominated
by that of the carbocation intermediates. Some terpene cyclases have been intensively investigated over
the past 10 years, and a good understanding of their mechanisms of action is starting to emerge. In some
cases, crystal structures of the enzymes are available, also with substrates/products or inhibitors bound at the
active site.

Before the emergence of gene cloning technologies, the study of terpene cyclases was very difficult,
because only very small amounts of enzyme could be isolated from natural sources. Far easier were
labelling experiments, in which 14C-labelled precursors were fed to intact plants. Several days to weeks
later, the natural products were isolated and examined to determine how the radioactive label had been
incorporated (if at all):

Me OH
1,8-Cineol

HO O Eucalyptus globulus
HO O
MVA
E !-Pinene
Pinus nigra

O
OPP
Thujone
Thuja occidentalis
GPP

(+)-Campher from
Salvia officianalis
(-)-Campher from Campher
Tanaceteum vulgare
O

The labelling experiments, if designed well, would provide insights into how the precursor GPP must fold
and cyclize to produce the natural product. The occurrence of intermediates in the pathway could be sought
in extracts of the plant. Over the past 10 years or so, with advances in molecular genetics and genomics,
direct access to the genes for the biosynthetic enzymes has been obtained. With recombinant DNA methods
the enzymes can be produced in large amounts and their mechanisms can be studied directly in vitro.

The monoterpene cyclases (see Chem. Rev. 1987, 87, 929) from plants have been well studied. These
enzymes use GPP as substrate, and catalyze typically a unique cyclization reaction. However, sometimes
more than one product is observed! The enzymes usually required Mg2+ or Mn2+ as cofactor.

One important question is: how can GPP (with an E-double bond) be cyclized to produce a 6-membered
ring? In a first step, the GPP is converted into an enzyme bound (3R)- oder (3S)-linalyl pyrophosphate,
which, after a change in conformation, then reacts further to cyclic products, e.g.
OPP OPP
OPP
OPP
Cyclase
 10
The resulting -terpinyl carbocation remains a bound intermediate in many terpene cyclase reactions, and
can react further in many different ways. Each monoterpene cyclase will typically catalyze preferentially
one reaction pathway:

OPP

(-)-Limonene
3-Carene
OH
!-Terpineol

Sabinene
Terpinolene

1,8-Cineol !-Thujene

!-Terpinyl-Kation
(+)-Bornyl-
pyrophosphate
OPP

"-Terpinene

Camphene
OH

!-Terpinene
!$Pinene
-Pinene
endo-Fenchol
#-Phellandrene

One well studied example is the bornyl pyrophosphate cyclase, which is involved in the biosynthesis of
camphor :

Bornyl-PP
OPP OPP
cyclase

O
OH
(+)-Camphor
(+)-Borneol

Mechanism of the cyclase reaction:

OPP OPP OPP

OPP OPP
H
HR H H H
HS H H H
Cyclase H

!
Bornyl
enzyme bound intermediates pyrophosphate
 11

Limonene synthase is another well-studied enzyme. (-)-Limonene is the precursor of menthol and carvone,
which can be isolated from extracts of peppermint, carraway (Carum carvi) and dill.

OPP
OH O O

(-)-trans-Isopiperitenol (-)-Isopiperitenon cis-Isopulegon

GPP (-)-Limonen

OH O O

(-)-Menthol (-)-Menthon (+)-Pulegon

The main product of the limonene synthase reaction is limonene, but small amounts of myrcene (2%), -
pinene und -pinene (4%) can also be detected:

OPP OPP
OPP
PPO
PPO

GPP
OPP OPP

-H
Myrcen
!-Pinen "-Pinen
(-)-4S-Limonen

Sesquiterpene Synthases (Curr. Opin. Struct. Biol. 1998, 8, 695; Chem. Rev. 1990, 90, 1089)
All sesquiterpenes are formed from FPP. A large variety of different cyclic sesquiterpenes have been
discovered in Nature.
!-Selinene
Germacrene C "-Humulene Me
H

!-Cadinene E--Farnesene O
H
O
O

O Me
O
Artemisinin

5-epi-aristolochen OPP
FPP

Amorpha-4,11-diene

Vetispiradiene E-#-Bisabolen
OH
epi-Cedrol
Trichodiene
Pentalenene
 12
The sesquiterpene cyclases require Mg2+ as cofactor and use FPP as substrate. The metal is coordinated
both to the protein and to the pyrophosphate group of the substrate. An important point is the stereochemical
course of the cyclization at C1, with some reactions proceeding with retention and others with inversion of
configuration. Mechanism in overview:

1,10 cyclization

- H+ Aristolochene
OPP OPP
FPHPH2 E--Farnesene

OPP
!-Humulene Longifolene
1,6- "-Ylangene
1,11-
-Bisabolene
1,10 cyclization

1,11 cyclization
OPP OPP
"-Longipinene

A well studied example is the enzyme trichodiene synthase from the fungus Fusarium sporotrichioides,
which converts FPP into trichodiene:

OPP OPP

OPP

H
H ! !
!

!
!
H
Trichodiene

The aristolochene synthase isolated from tobacco plants and the vetispiradiene synthase from Hyoscyamus
muticus are two phylogenetically closely related enzymes that catalyze also very closely related reactions. In
both cases, E,E-germacrene-A is formed as a short-lived enzyme-bound intermediate. Studies reported so
 13
far suggest the following mechanism of action:

5-epi-Aristolochene
Synthase
Vetispiradiene
Synthase

OPP OPHPH2
E,E-Germacradienyl cation Germacrene-A
FPP
+H

Vetispiradiene Aristolochene

Two other very interesting sesquiterpene cyclases are -humulene synthase und -selinene synthase, both
isolated from fir trees, which can catalyze the formation of a surprisingly large variety of cyclic
sesquiterpenes starting from FPP. In in vitro assays monitored by GC, the formation of 34 different products
catalyzed by -selinene synthase can be observed starting from FPP, whereas -humulene synthase catalyzes
the formation of 52 different products, although about half so far have unknown structures. Interestingly, the
same mixture of terpenoid products can also be found in fir trees (J. Biol. Chem. 1998, 273, 2078). See
below.

Diterpene Synthases.
The diterpene synthases catalyze similar reactions, but use now GGPP as substrate. The same principles
of reactivity apply. Thus the allylic pyrophosphate acts as a leaving group (with assistance by Mg2+), and the
resulting carbocation can initiate a variety of different reaction paths (carbocation-addition to double bonds,
rearrangements (Wagner-Meerwein), hydride shifts, as well as deprotonations) depending upon the bound
conformation at the active site of each enzyme. Examples are also seen where the protonation of a double
bond is used to initiate a cyclization reaction. A great structural diversity is seen amongst diterpene natural
products :

(-)-Kaurene
(-)-Abietadiene

OPP OPP OPP

GGPP

(+)-Copalyl Diphosphate (-)-Copalyl Diphosphate

Casbene
Taxadiene
 14
Taxol is an important natural product because of its anti-cancer activity. It was discovered in a National
Cancer Institute program at the Research Triangle Institute in 1967 when it was isolated from the bark of the
Pacific yew tree, Taxus brevifolia and named 'taxol'. When developed commercially by Bristol-Myers
Squibb (BMS) the generic name was changed to 'paclitaxel'. The BMS compound is sold under the
trademark 'Taxol'. Paclitaxel is now used to treat patients with lung, ovarian, breast cancer, head and neck
cancer, and advanced forms of Kaposi's sarcoma. Paclitaxel is also used for the prevention of restenosis.
Paclitaxel works by interfering with normal microtubule growth during cell division.

From 1967 to 1993, almost all the paclitaxel produced was derived from the bark of the Pacific yew, the
harvesting of which kills the tree in the process. In 1992 BMS started to manufacture paclitaxel from 10-
deacetylbaccatin isolated from the needles of the European yew. By the end of 1995, BMS stopped
production from the bark of the Pacific yew, effectively terminating the ecological controversy over its use.
Currently, all paclitaxel production for BMS uses plant cell fermentation technology. This starts from a
specific taxus cell line propagated in aqueous medium in large fermentation tanks. Paclitaxel is then
extracted directly, purified by chromatography and isolated by crystallization.

There is now great interest in trying to reconstitute the entire biosynthetic pathway in vitro. Several of the
enzymes on the pathway have already been cloned and produced by recombinant DNA techniques. A key
step is catalyzed by the taxadiene synthase:

Me O O OH
O Me
Me
Me
O O
GGPP Me
N O H O
H O O
OH HO Me
O O
Taxol Ph

The mechanism of the cyclization has been intensively studied:

D H
D

OPP H
D
D

H
H

2.6. The formation of triterpenes from squalene (Angew. Chem. 2000, 112, 2930)

Squalene is the universal precursor of all triterpenes, including all steroids. In animals, squalene is
converted in only two steps into a steroid called lanosterol. The first step is catalyzed by a monooxygenase,
which is a flavo-enzyme not a hemoprotein, but uses molecular oxygen and NADPH to epoxidize squalene:
 15
Me Me Me
Me
Me Squalene
Me Me Me
NADPH, O2
NADP+, H2O squalene epoxidase
H
Me Me Me
Me Me
squalene epoxide H Me
Me
cyclase Me
Me Steroide
Me
Me
Me HO
Squalene Epoxide Me Me Lanosterol
O Me
Me

Perhaps of most interest here is how the cyclase enzyme can take squalene epoxide as substrate and release
lanosterol as product. What chemical steps take place at the active site of the enzyme and how is the
reaction catalyzed?

Oxidosqualene-Lanosterol-Cyclase
In higher organisms the steroid skeleton is produced through the action of a membrane-bound enzyme. In
the course of the transformation, a series of ring-forming steps and rearrangement reactions take place:

Me H O Me
Me Me
X=O
Enzyme HO H
Me
BH Me X H Me H
Me Me
O
Me Me
Me Me
Me H Me
Me Me
HO H
Me Me
H H
Me
Me
Me H Me
Me
H
HO
Me Me Me ?
Me H
Me H Me
Me Me Me
HO H Me
Me Me H Me
H Me H Me Me
Me H
Me HO
Me Me Me
H H
Me
Me H Me
Me H Me
Me Me
H
HO Me H
Me
Me H Me
Me
H
HO
Me
Me H
Br O
N Lanosterol

O F Ro 48-8071
an inhibitor
 16
The cyclase must bind the substrate in the correct folded conformation to allow a stereoelectronically
assisted series of rapid ring closure steps, and form the product with the correct relative and absolute
configuration. All the intermediate carbocation intermediates must be shielded from reaction either with
water or with the protein. Finally, the correct proton must be removed to terminate the reaction. The H-atom
shifts and Wagner-Meerwein rearrangements occur along a kinetically and thermodynamically preferred
pathway, until the end product is reached. At the end of 2004 a group at Hoffmann-La Roche in Basel
succeeded for the first time in crystallizing the enzyme (Nature, 2004, 432, 118).

Left: Ribbon diagram of human OSC. a, The C and N termini and several sequence positions are labelled. The
inner barrel helices are coloured yellow. The bound inhibitor (black) indicates the location of the active site. b,
The orientation of OSC relative to one leaflet of the membrane, whose polar and nonpolar parts are depicted in
light blue and light yellow respectively. Internal surfaces and channels of OSC are shown with the following
colour code: blue, positive; red, negative; cyan, hydrogen-bond donor; magenta, other polar. Ro 48-8071 binds in
the central active-site cavity. Two channels lead to the enzyme surface: one is hydrophobic to the membrane
insertion site and one is polar. The fragment of lipid (blue) binds to the hydrophobic substrate entrance
channel. A -OG molecule belonging to a crystal neighbour (black) interacts with the membrane-inserting
hydrophobic surface.

Right: Stereoview of the electron density representing the bound substrate. Residues in the enzyme within 5
are shown. A/B-Rings: The cationic intermediates may be stabilized by cation- interactions with the aromatic
rings of Trp387, Phe444 and Trp581. The catalytic Asp455 is activated by Cys 456 and Cys 533. The Tyr 98
side chain sterically hinders the B-ring from assuming the favourable chair conformation. C/D-Rings: Phe 696
and His232 can stabilize the positive charge at the C20 cation by cation- interactions. His 232 is the nearest
basic residue that could deprotonate the C8/9 lanosterol cation.
 17
From lanosterol, the pathway for steroid biosynthesis continues on to cholesterol. Three methyl groups must
be removed, one double bond is reduced and another is shifted. Cholesterol then becomes a branch point in
steroid biosynthesis, serving as a precursor from which other steroids are produced:

#
!" !" #
!" !"
!"
# !" !"
# !"
!"
!" #
!" # #
#$
Lanosterol #$ Cholesterol
!" !"

In plants the oxidosqualene cyclase does not form lanosterol, but rather cycloartenol, which is then the
precursor for the formation of other plant steroids:

Me
Me
Me Me H Me
Me Me Me Me
BH Me
Me
O Me HO H
Me Me
Me Me H H

Me
Me
Me
Me
Me
Me Me H
Me H
Cycloartenol Me H
Me
Me H
H HO
HO Me
Me Me H
Me H

Bacterial squalene cyclase catalyzes a different cyclization cascade, which is mechanistically related, but
not so complicated. Now squalene (not the epoxide) is bound in a specific conformation, which allows a
rapid series of cyclization steps to occur. The process is now started by protonation of the terminal double
bond (Chem.Biol. 2000, 7, 643):

Squalene-Hopene
Cyclase

This cyclase is a homodimeric, soluble enzyme. The active site is a buried cavity, which binds squalene in
the preferred conformation. Most probably the side chain of Asp376 acts as a general acid catalyst to start
the cyclization cascade.
18
19