FEMS Microbiology Letters 13 (1982) 131-135
Published by Elsevier Biomedical Press
131
Hypothesis
Considerations on the evolution of steroids as membrane
components
Karl Poralla
lnstitut fi~r Biologie IL Lehrbereich Mikrobiologie L Universit& Tikbingen, A uf der Morgenstelle 28, D- 7400 Ti~hingen, F. R. G.
Received 14 September 1981
Accepted 20 September 1981
1. O X Y G E N A S E R E A C T I O N S
SYNTHESIS OF STEROIDS
AND
BIO-
Metabolic pathways depending on molecular
oxygen introduced by oxygenases to organic molecules are, for two reasons, presumably late inventions of evolution. The primordial atmosphere of
the earth was nearly devoid of molecular oxygen
according to several models [1]. After the invention of the oxygenic photosynthesis, oxygen concentration in the atmosphere rose after a lag period
very slowly. According to a hypothesis of Schidlowski, an oxygen content of 0.2% in the atmosphere was reached two billion years ago [2]. Presumably this low oxygen content is not sufficient
to drive oxygenase reactions. A second argument
for the late invention of oxygenase reactions is
their complete absence in amino acid and nucleotide biosynthesis, and in the first steps of other
biosynthetic pathways. Oxygenase reactions occur
in the last steps of biosynthetic pathways of unsaturated fatty acids in most aerobic organisms,
steroids, and xanthophylls. The initial reactions in
the catabolism of hydrocarbons, aromatic compounds, and amino acids are comprised by
oxygenases. An example of a catabolic reaction
functioning as a biosynthetic one is the hydroxylation of phenylalanine to tyrosine in animals.
A pathway which is anaerobic in the first part
and dependent on molecular oxygen in the second
Epoxysqualene
In animals
fungi
and
J
Lanosterol
Cholesterol
/
~
In photosynx,x~etic plants
Cyc[o art enol
Ergosterol etc
Fig. 1. The two cyclization reactions for the synthesis of steroids
in eukaryotes. Some organisms can convert cycloartenol to
lanosterol, without synthesizing the former molecule. In fungi
the pathway starts from lanosterol; in photosynthesizing plants
ergosterol, sitosterol, and similar steroids are synthesized starting from cycloartenol [4]. But in red algae cycloartenol is the
precursor for the main steroid cholesterol [3].
0378-1097/82/0000-0000/$02.75 c, 1982 Federation of European Microbiological Societies
132
L o n o s t e r o[
Cholesterol
Fig. 2. An example of the mammalian route for cholesterol
synthesis. The drawn steps do not correspond with the number
of reactions. For each demethylation five or six reactions are
necessary. In total 19-20 reactions are required for processing
lanosterol to cholesterol. Steps 2, 4, and 5 are dependent on
molecular oxygen [4].
part, is the biosynthesis of steroids. The first part
starts from acetyl-CoA via mevalonate and farnesyl pyrophosphate to squalene. Then an oxygenase
converts squalene to epoxysqualene thereby consuming molecular oxygen. In animals and fungi
this compound is cyclized to lanosterol, and in
photosynthetic plants to cycloartenol (Fig. 1) [3].
At least three molecules of oxygen are consumed
in the conversion of lanosterol to cholesterol.
About 19 reactions (Fig. 2) are necessary for
processing the cyclization products lanosterol and
cycloartenol to the final membrane components
cholesterol or ergosterol [4]. Thereby molecules are
synthesized which are free of substituents at the
a-site.
Because molecular oxygen is a substrate for
several reactions in the biosynthesis of sterols,
yeast cells cannot grow anaerobically in the absence of appropriate sterols [5].
2. T H E F U N C T I O N S OF S T E R O I D S
In higher organisms steroids fulfill different
functions. First of all they occur in cellular mere-
branes of nearly all eukaryotes. The most important membrane steroid is cholesterol. This compound is the biosynthetic precursor of bile acids
and steroidal hormones. Steroids can also occur as
esters and glycosides. I will confine the discussion
on evolution to steroids as membrane components.
It is possible that some selection pressure on the
processing reactions has its origin from the
hormonal functions of the steroids. But in protozoa and yeasts steroids exist solely as membrane
components. So one can at least for these groups
exclude a selection pressure originating from
hormone function.
As membrane components steroids have a distinct function which is not yet fully understood.
Mainly experiments with artificial lipid membranes revealed that depending on the concentration cholesterol diminishes or abolishes phase
transitions of glycerolipids. The fluid lipid phase is
condensed by sterols, so that the mobility of the
acyl-chains of lipids is restricted. In contrast the
gel phase of a lipid membrane becomes more fluid
by cholesterol and similar sterols [6]. The structural requirements of the sterol molecule for membrane function are more rigid in systems of higher
organisms than in bacterial systems. The membrane sterol in eukaryotes contains no 4-methyl
group. Whereas the methylotrophic bacteria Methylococcus [7] and Methylobacterium [8] contain
sterols which are methylated at the C4-position.
But the function of these sterols is not yet studied.
Furthermore, in the cell-wall-less bacterium
Mycoplasma the cyclization products lanosterol
and cycloartenol are growth promoting [9]. Both
compounds possess 4- and 14-methyl groups.
Two possible properties of sterols in membranes are rarely discussed. Uncharged sterols are
suitable agents for separation and thereby lowering repulsion of charged head groups of lipids [10].
As a consequence, a membrane must gain more
stability. Secondly a bilayer alters its thickness
depending on temperature [11]. If rigid molecules
are inserted, temperature-dependent alterations of
a bilayer thickness will be reduced. This can be of
importance of membrane function, especially for
some electrical phenomena.
133
3. T H E E V O L U T I O N OF STEROIDS A N D
HOPANOIDS
An interesting question accompanying biosynthesis and function of steroids is the evolution
of their biosynthetic pathway. Thereby one can
consider, and eventually elaborate general evolutionary features also of other biosynthetic pathways. I will review and discuss a few ideas of the
evolution of steroidal biosynthesis. Considerations
on the evolution of steroids especially after the
cyclization reaction were first raised by Nes [12]
and Bloch [13]. The early paper of Nes was mainly
concerned with the alkylation step at C24 in the
side chain. This alkylation is typical for steroids in
fungi and photosynthetic plants. But the importance for membrane function is not yet understood. Bloch studied precursors of cholesterol and
ergosterol in membranes of Mycoplasma and a
sterol requiring yeast mutant [9,14]. Both primary
cyclization products, cycloartenol and lanosterol,
show significant growth promotion and enhance
microviscosity in membranes of Mycoplasma.
Therefore, further processing of the cyclization
products seems only necessary for the improvement of membrane function of the steroids. The
question of anaerobically synthesized structural
~
Hopene
~
/
" ]~'OH '
Diplopt erol
OH
" ~ ~ O H
OH
OH
",
Tetrahydr oxy bact e r i o h o p a n e
Fig. 3. Examples of hopanoids in bacteria. Hopene and diplopterol are primary cyclization products from squalene. Tetrahydroxybacteriohopane is a 'processed' hopanoid. The hydroxylated sidechain can further be substituted by glucosamine
[IS,161.
equivalents, and of phylogenetic precursors of
steroids was first rigorously taken into account by
Ourisson et al. [15,16]. These investigators proposed the idea that hopanoids (Fig. 3) and other
terpenoids could be candidates for the abovementioned roles. They argued that hopanoids are
phylogenetic precursors of steroids because of their
similar structure to steroids, their anaerobic biosynthesis, the simple cyclization reaction of squalene without shifting of methyl groups, wide distribution in prokaryotes, and their ubiquitous occurrence in geological sediments and crude oil up to
an age of 5. l0 s years. Furthermore, hopanoids
and cholesterol condense monolayer membranes.
In this system both cyclic terpenoids show a condensing effect and abolish phase transition [ 17,18].
4. T H E P O S I T I O N O F
STEROID EVOLUTION
SQUALENE
IN
I want to include squalene as a possible phylogenetic precursor of steroids. At low concentrations squalene can well be an advantageous component in the cytoplasmic membrane. First, it
could be of value as an agent to fill free lattice
space in the membrane. It is not per se clear that a
biological membrane is built in such a perfect
manner that all components fit exactly together.
For example, insertion of proteins into a lipid
membrane may involve formation of free lattice
space. Second, an apolar component as squalene
together with uncharged lipids could separate
molecules with charged headgroups. Therefore,
squalene could stabilize certain types of membranes.
The function of squalene was first studied by
Lanyi et al. [19]. These results obtained with
Halobacterium membranes suggested that squalene
projects into both halves of the bilayer, perpendicularly to the plane of the membrane, and occupies
some of the free lattice space. Furthermore, squalene was used as a bulk phase in black lipid
membranes [20]. This experiment shows that
squalene is able to fill up the critical region where
the bilayer passes into the torus, used in this
technique. This is a good indication for the potential of squalene for filling out even big cavities.
134
The wide occurrence of squalene in bacteria is as
yet not functionally understood. Out of 73 Grampositive bacteria tested, 65 possess squalene [21].
Many of these bacteria are not known to contain
steroids, hopanoids, C30-carotenoids or other
potential derivatives of squalene. Therefore, a
function as a biogenetic precursor can be excluded. In these cases squalene may fulfill a membrane function in the above-mentioned manner.
The arguments for squalene as a potential membrane component are also valid for epoxysqualene.
It is conceivable that this biosynthetic steroid precursor possesses a more parallel orientation to the
acyl chains of the accompanying lipids by virtue of
the polar epoxy group.
5. T H E POSITION OF
STEROID E V O L U T I O N
HOPANOIDS
IN
During the course of evolution of organisms,
advantages could be gained for a lipid membrane
by the formation of a squalene cyclase which
synthesized a polar, polycyclic and flat molecule.
The main advantage of such a molecule is the
fixed conformation of the ring system in contrast
to the variable, temperature-dependent conformation of the acyl chains in the glycerolipids. Hypothetical candidates for such molecules are the
hopanoids (Fig. 3) as proposed by Ourisson and
coworkers [15,16]. The rigid and uncharged
hopanoids can fulfill the function of separation of
charged lipids and simultaneously gain new functions in the reinforcement of the membrane above
the melting point of the glycerolipids.
For unknown reasons hopanoids or similar
cyclization products of squalene do not occur as
components in membranes of eukaryotes. The only
known exception of this rule is the tetrahymanol
in a ciliate. It is conceivable that the conformation
of a pentacyclic molecule without an apolar side
chain is too rigid in contrast to the tetracyclic
steroids with a fairly long side chain. Furthermore
hopanoids are not processed at the nucleus; in
contrast the side chain is elongated and hydroxylated (Fig. 3). In this context hopanoids and other
pentacyclic triterpenes can be considered as a side
branch in the evolution of steroids. But it is still
possible that the squalene cyclase further evolved,
for example starting from a gene duplication, to an
epoxysqualene cyclase synthesizing a steroid [16].
This hypothesis can be clarified by comparing the
amino acid sequence of both enzymes. The sequence analysis could be the basis for deciding the
alternative: are the cyclases homologous, or enzymes with convergent evolution?
Some indications for the course of evolution of
the steroidal biosynthetic pathway can be gained
by comparing intermediary products in their membrane properties. According to a hypothesis of
Bloch [10] the first steroidal cyclization products
were further stepwise processed to yield a molecule
highly adapted to interaction with glycerolipids
containing unsaturated fatty acids in the C 2position of the glycerol. It is interesting that the
first demethylation-step in biosynthesis in mammalian and yeast cells takes place at C~4. This
methyl group protrudes from the otherwise planar
a-face and therefore disturbs the interaction with
glycerolipids [22]. One of the last steps in the
biosynthesis are the two demethylations at C 4.
Possibly the methyl groups at C 4 can sterically
hinder the hydroxy group at C 3 in the formation
of a hydrogen bond according to a hypothetically
model of Huang [23].
6. D I F F E R E N T TYPES OF EVOLUTION OF
BIOSYNTHETIC PATHWAYS
The evolution and biosynthesis of cholesterol
and ergosterol can be hypothetically envisaged as
a stepwise improvement of a membrane component starting from squalene via epoxysqualene and
the first cyclization products. All intermediary
products starting from squalene, had a membrane
function as is partly shown for the steroidal part
of the pathway by Bloch [9,14]. This evolutionary
new steroid pathway could have evolved by a type
of forward-evolution. Perhaps for all metabolites
not occurring in the prebiotic organic soup this
type of evolution can be envisaged. For the pathways of molecules which occurred in the prebiotic
soup Horowitz [24] made the proposal of a backward evolution. After consumption of a product in
the prebiotic ocean an enzyme has evolved which
135
converts a prebiotic precursor to the product. In
this way a net of catalytic reactions was created
until the biosynthetic pathways were connected
with the central energy creating pathways (glycolysis and citric acid cycle). In this type of backwardevolution of a biosynthetic pathway evolution is
guided by the occurrence of the precursors in the
primordial soup. In forward-evolution the process
is guided by the partial fulfillment of function of
the precursors, which gains a stepwise improvement. This view of evolution is a selectionistic one.
It is possible that in other biosynthetic routes, for
example in secondary metabolism a neutralistic
view (playground-hypothesis) is more justified [25].
ACKNOWLEDGEMENTS
I want to thank Dr. H. Anke for critical reading
the article and L. Curnick-Cole for improving the
English.
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