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INTRODUCTION |
Chloroflexus aurantiacus is considered as the most
basal branch of a photosynthetic organism in eubacterial phylogeny
which has been described hitherto (1-3). The bacterium shows unusual characteristics. The fixation of CO2 occurs via a novel
cyclic pathway discovered recently (4, 5). The primary step of this
pathway is the carboxylation of acetyl-CoA. The resulting malonyl-CoA
yields hydroxypropionate, which is converted to propionyl-CoA by a
sequence of dehydration and reduction reactions. A further carboxylation reaction of propionyl-CoA leads to dicarboxylic acids,
which may be metabolized further to acetyl-CoA and glyoxylate (5).
The cell wall and membrane composition of C. aurantiacus are
exceptional among procaryotic organisms. Membrane phospholipids are
characterized by the predominance of saturated and unsaturated C18 and C20 fatty acids besides unusual fatty
acid residues with a chain length of 17 (6).
Monogalactosyldiglyceride, sulfoquinovosyldiglyceride, and most
notably, phosphatidylinositol and diglycosyldiglycerides are found
beside phosphatidylglycerol. The glycolipids contain equimolar amounts
of glucose and galactose (6, 7). Moreover, the lack of
lipopolysaccharides is characteristic for the unusual cell membrane
composition (8). L-Ornithine is the only diamino acid of
the peptidoglycan. Instead of protein, a complex polysaccharide is
bound to the peptidoglycan (9). The carotinoid composition is also
different from that of other procaryotes (10).
The diterpene verrucosan-2
-ol (compound 1, Fig.
1) has been isolated recently by Hefter
et al. (11) from the membrane fraction of C. aurantiacus in yields of 1-2 mg from a 1-g cell mass (dry
weight). Previously, this rare diterpene with an unusual 3,6,6,5-tetracyclic ring system had been described only in marine sponges (12) and some liverworts (hepaticae) (13-21), which are considered as early members of the evolution of terrestrial plants. Hopanoids and steroids have not been found in the membrane of C. aurantiacus. Despite its different molecular dimensions, it has
been proposed that verrucosan-2
-ol can act as a modulator of
membrane fluidity in analogy to hopanoids and steroids in other microorganisms (11).

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Fig. 1.
IUPAC conventions for verrucosan-2 -ol
(1), isopentenyl pyrophosphate (2), acetyl-CoA
(3), pyruvate (4), and glyceraldehyde
3-phosphate (5).
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Current evidence suggests that isopentenyl pyrophosphate
(IPP)1 and dimethylallyl
pyrophosphate (DMAPP) act as the universal precursors of terpenoid
biosynthesis. The two precursors can be interconverted by isopentenyl
pyrophosphate isomerase. The biosynthesis of cholesterol from acetate
via mevalonate has been studied in detail by Bloch, Lynen, and their
co-workers (22). More recently, a second pathway via
1-deoxy-D-xylulose or its 5-phosphate has been discovered
in plants and in certain eubacteria by independent work of the groups
of Arigoni and Rohmer. More specifically, Arigoni and co-workers
reported that 1-deoxyxylulose can serve as precursor for quinoid
coenzymes in Escherichia coli and for ginkgolides in
Ginkgo biloba. On the other hand, these authors found that sterols are formed in Ginkgo via mevalonate (23-25). Sahm,
Rohmer, and their co-workers found that the biosynthesis of hopanoids in the eubacterium Zymomonas mobilis and ubiquinone Q8 in
Escherichia coli proceed via the deoxyxylulose pathway (26,
27). Furthermore, the deoxyxylulose pathway has been shown to be
operative in the alga Scenedesmus obliquus (28), in cell
cultures of the plants Taxus chinensis (29),
Catharanthus roseus (30), Rauwolfia serpentina,
and Rubia tinctorum (31), and in the plants Lemna gibba, Hordeum vulgare, Daucus carota (32),
and Mentha piperita (33).
The pathway of terpenoid biosynthesis in C. aurantiacus is
unknown. Moreover, the biosynthesis of verrucosan type compounds has
not been studied in any species to the best of our knowledge. In order
to study the biosynthesis of verrucosan-2
-ol, we applied singly and
doubly 13C-labeled acetates to growing cultures of C. aurantiacus. The C enrichments and the
13C-13C coupling patterns were determined by
one- and two-dimensional C and 1H NMR
spectroscopy. The labeling pattern of cellular amino acids and
nucleosides served as a basis for the reconstruction of central metabolic pools by retrobiosynthetic analysis (5). These data clearly
show that IPP and DMAPP are formed by the mevalonate pathway in
C. aurantiacus. A specific cyclization mechanism for the
formation of verrucosan type diterpenes is proposed.
 |
EXPERIMENTAL PROCEDURES |
Chemicals--
[1-13C]Acetate and
[2-13C]acetate were purchased from MSD-Isotopes
(IC-Chemikalien, München, Germany) and
[1,2-13C2]acetate from Isotec (Miamisburg,
OH). Gases were purchased from Linde AG (Höllriegelskreuth,
Germany).
Microorganism--
C. aurantiacus (DSM 636) was
obtained from the Deutsche Sammlung von Mikroorganismen
(Braunschweig, Germany). Growth conditions and culture media have been
described earlier (34).
Isotope Incorporation Studies--
C. aurantiacus was
grown phototrophically under anaerobic conditions as described earlier
(34). The feeding experiments with [1-13C]acetate or
[2-13C]acetate (99% 13C) have been described
(5). Briefly, the sodium salt of the respective 13C-labeled
acetate was added as a sterile solution to a final concentration of 10 mM when the culture had reached a value of 30-100 mg of cell protein per liter.
An experiment with [1,2-13C2]acetate was
performed with continuous feeding. A sterile solution (32 ml)
containing 3.44 mmol of sodium [1,
2-13C2]acetate, 18.6 mmol of unlabeled sodium
acetate, and 2.4 µCi of [1,2-14C]acetate was added to
the growing culture (2 liters) during a period of 100 h using a
peristaltic pump (Braun, Melsungen, Germany). Aliquots were retrieved
at intervals, and cell protein was determined by a modified Lowry
method (35). The feeding rate was adjusted proportional to the growth
rate. The radioactivity of cells and the supernatant culture medium was
monitored by liquid scintillation counting with external
standardization. Cells were harvested aerobically when the solution of
labeled acetate was consumed.
Isolation of Verrucosan-2
-ol--
Bacterial cell mass (0.9 g
dry weight) from the growth experiments with
[1-13C]acetate or [2-13C]acetate was
freeze-dried and suspended in a mixture of ethanol/diethylether (3:1,
v/v, 200 ml). The suspension was boiled under reflux for 90 min. Solids
were removed by filtration, and the solvent was evaporated under
reduced pressure. The residue was dissolved in 20 ml of a
hexane/dichloromethane mixture (1:1, v/v). The solution was placed on a
column of Merck Silica Gel 40 (4 × 30 cm). The column was
developed with 1000 ml of hexane followed by 1200 ml of dichloromethane
and by a mixture (300 ml) of methanol/dichloromethane/water (6:3:1,
v/v). Fractions of 30 ml were collected. Verrucosan-2
-ol (1.1 mg)
was eluted after 500 ml of dichloromethane and was identified by
1H NMR spectroscopy.
Cells grown with [1,2-13C]acetate (1.4 g dry weight) were
suspended in a mixture of methanol/dichloromethane (1:1, v/v, 200 ml).
The mixture was ultrasonically treated for 15 min until the supernatant
was colorless. Chromatography on silica gel was performed as described
above.
The fractions from the experiment with
[1,2-13C2]acetate containing
verrucosan-2
-ol were subjected to solid phase extraction using a
cartridge of C18 reversed phase material (Accu Bond, 5 ml)
and methanol as eluent. The eluate was further purified by preparative
thin layer chromatography using a Silica Gel 60 TLC plate (Merck) and a
mixture of hexane/dichloromethane (6:4, v/v) as solvent.
Verrucosan-2
-ol had an RF value of 0.78. The
band containing the diterpene was scraped off. The solid was extracted with 30 ml of dichloromethane for 30 min. Evaporation of
dichloromethane under reduced pressure yielded 1.3 mg of
verrucosan-2
-ol. The isolation of amino acids and nucleosides has
been described earlier (5, 36).
NMR Spectroscopy--
1H and 13C NMR
spectra were recorded at 500 and 125 MHz, respectively, using a Bruker
DRX 500 NMR spectrometer. One-dimensional and two-dimensional
experiments (COSY, NOESY, INADEQUATE, HMQC, and HMBC) were performed at
8 °C using standard Bruker software (XWINNMR).
Quantitative Analysis of 13C
Enrichment--
Absolute 13C abundance was determined for
each carbon atom of verrucosan-2
-ol obtained from the feeding
experiments by quantitative NMR spectroscopy (34). Briefly,
1H decoupled 13C NMR spectra of biosynthetic
samples and of samples with natural C abundance (1.1%
13C) were measured under identical conditions. Relative
13C abundance of individual carbon atoms was then
calculated from the integrals of biosynthetic samples by comparison
with the natural abundance samples. In order to obtain absolute
13C enrichments, the 13C satellites in the
1H NMR of each metabolite were analyzed yielding absolute
13C-enrichment values for selected carbon atoms. On the
basis of these data, the relative 13C abundances were then
referenced to absolute values.
Quantitative Analysis of 13C-13C
Coupling--
The fraction of multiply labeled isotopomers was
calculated from 1H-decoupled 13C NMR spectra as
the fraction of 13C-13C coupled satellites
relative to the integral of the entire 13C signal of the
respective carbon atom.
NMR Spectra Simulation--
13C-Coupling patterns
were simulated with NMRSIM software (Bruker).
NOE Studies--
Build-up rates of NOEs were determined by
two-dimensional NOESY experiments using mixing times of 20 ms to 3 s. Interproton distances were calculated by standardization on the
distance of the geminal protons at C-6 and C-12 estimated as 1.78 Å.
Molecular dynamic simulation of verrucosan-2
-ol was performed using
interproton distances from NOESY experiments and a simulated annealing
protocol. Energy minimization was performed using the MM+
force field and the software package DISCOVER (Biosym Inc.).
 |
RESULTS |
A prerequisite for the interpretation of biosynthetic tracer
studies by quantitative NMR spectroscopy is the unequivocal assignment of all NMR signals of the target molecule. A 1H and
13C NMR analysis of verrucosan-2
-ol with
CD2Cl2 as solvent has been reported by Hefter
et al. (11). Our initial NMR analysis using
CDCl3 as solvent gave some ambiguities with respect to the published assignments. It was therefore in order to assign all H and 13C NMR signals of verrucosan-2
-ol in
CDCl3 by two-dimensional NMR techniques. The 1H
NMR signals were analyzed by two-dimensional COSY and NOESY type
experiments (Table I, Fig.
2). The 13C NMR signals were
assigned on the basis of DEPT and two-dimensional HMQC and HMBC
experiments (Table I). Additional information was gleaned from
two-dimensional INADEQUATE experiments using 13C-labeled
samples isolated from the growth experiments with
13C-labeled acetate (see below). With the exception of C-3
and C-16 which had to be interchanged, the data were in line with those obtained in CD2Cl2 by Hefter et al.
(11).
Interproton distances were calculated from the initial rate of NOE
build-up measurements as described under "Experimental Procedures"
(Table II). The conformation of
verrucosan-2
-ol was analyzed by molecular dynamics simulation. The
preferred conformation is shown in Fig.
3. The Re-methyl group (C-17)
is projected between C-14 and C-12 of the five-membered ring. Both the
observed upfield shift of C-17 caused by two
effects and the
coupling constant of 2.6 Hz between H-13 and H-15 are in keeping with
the conformation shown in Fig. 3. It should be noted that a similar
conformation was found in the crystal structures of
2
,9
-dihydroxyverrucosane and 2
,8
-dihydroxyverrucosane from
liverwort species (15, 18).
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Table II
Interproton distances (in Å) calculated from NOE initial rates and
from energy minimization structure by molecular dynamic methods
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Fig. 3.
Newman projection of the conformation of
verrucosan-2 -ol along the C-15/C-13 bond. This conformation was
gleaned from the analysis of NOE initial rates and molecular dynamics
calculations.
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Growth experiments with [1-13C]acetate and
[2-13C]acetate have been reported earlier (5). Briefly,
820 mg (10 mmol) of 13C-labeled acetate and a tracer amount
of 14C-labeled acetate (100 kBq) were added as a bolus to a
bacterial culture (1 liter) growing phototrophically on a mixture of
H2/CO2 (80:20, v/v). The cell mass was
extracted with ethanol/ether, and verrucosan-2
-ol was isolated from
the supernatant as described under "Experimental Procedures."
From the solvent-extracted biomass, amino acids and ribonucleosides
were obtained after hydrolysis of protein and RNA, and their
13C-labeling patterns were determined by NMR spectroscopy.
These data were used to reconstruct the labeling patterns of
intermediary metabolites such as acetyl-CoA (compound 3,
Fig. 1) and pyruvate (compound 4, Fig. 1). Results are shown
in Fig. 4 and Table
III. Some of these data have been
reported earlier (5). The labeling pattern of glyceraldehyde
3-phosphate (compound 5, Fig. 1) was deduced from C-3 to C-5
of ribose as determined from isolated nucleosides. We have shown
repeatedly that C-3 to C-5 of the pentose pool reflect the labeling
pattern of C-1 to C-3 of the triose phosphate pool in a variety of
organisms (5, 34, 36). The reconstructed 13C enrichments
for each carbon atom are shown as bold numbers (percent C abundance) in Fig. 4. The quantitative contributions of
satellite signals originating from coupling via a single bond to an
adjacent C atom are indicated by italic
numbers and arrows. The arrows point to the observed
carbon atoms, and the numbers indicate the fraction of the coupled
species referenced to the total signal integral of the observed
carbon.

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Fig. 4.
Averaged labeling patterns of acetyl-CoA,
pyruvate, and glyceraldehyde 3-phosphate. Absolute 13C
enrichments (%) are shown as bold numbers.
13C-13C coupling determined from the fraction
(%) of coupling satellites relative to the entire signal integral is
shown in italics. The arrow points to the carbon
atom which shows the coupling ratio specified by the italicized
number. Data for experiments with [1-13C]- and
[2-13C]acetate are from Eisenreich et al.
(5).
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Table III
13C enrichments of verrucosan-2 -ol and central metabolites
from C. aurantiacus supplemented with 13C-labeled acetates
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It should be noted that coupling cannot be predicted from enrichment in
this study. For example, in pyruvate derived from [1-13C]acetate, the coupling contributions are low
despite the enrichment values of 19-28% of the carbon atoms. This
reflects the fact that the biosynthetic material is an isotopomeric
mixture of [1-13C]-, [2-13C]-, and
[3-13C]pyruvate. The formation of the single-labeled
isotopomers by the carbon fixation cycle (as opposed to a stochastic
distribution of 13C atoms which would be conducive to the
formation of a substantial fraction of double-labeled isotopomers)
has been discussed earlier (5).
The bolus addition of [1-13C]- and
[2-13C]acetate shifted the growth pattern of the culture
to a heterotrophic mode with efficient acetate utilization (5). The
resulting high 13C enrichment of central intermediary
metabolites such as acetyl CoA and pyruvate was favorable for the
analysis of the terpene cyclization (for details see below). On the
other hand, flooding of the acetyl-CoA pool with
[1,2-13C2]acetate would have been conducive
to the formation of totally labeled verrucosan-2
-ol devoid of
meaningful biosynthetic information. A growth experiment was therefore
performed with slow, continuous addition of
[1,2-13C2]acetate to the growing culture over
a period of 100 h. Experimental details are described under
"Experimental Procedures." Under these conditions, the proffered
acetate is diluted by material obtained from phototrophic
CO2 fixation.
Absolute 13C abundance data for verrucosan-2
-ol from
growth experiments with 13C-labeled acetates were
determined by 1H and 13C NMR spectroscopy as
described under "Experimental Procedures." Parts of NMR spectra are
shown in Fig. 5. 13C
enrichments are summarized in Table IV
and Fig. 6.

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Fig. 5.
Part of 13C NMR spectra of
verrucosan-2 -ol. 13C-13C-Coupling
patterns as identified by INADEQUATE experiments are indicated on
top of the spectra. A, natural 13C
abundance; B, from [1-13C]acetate;
C, from [2-13C]acetate; D, from
[1,2-13C2]acetate.
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Table IV
13C Enrichments in verrucosan-2 -ol isolated from cells of C
aurantiacus grown for several generations with CO2 (natural
13C abundance) in the presence of 13C-labeled acetate
[1-13C]Acetate (99% enrichment) or [2-13C]acetate
(99% enrichment) were added as bolus to a concentration of 10 mM. A mixture of [1,2-13C2]acetate and
unlabeled acetate at a molar ratio of 1:4 was added continuously over a
period of 100 h. The amount of acetate added was 22 mmol per
liter. The percentage coupling is the fraction of
13C-13C coupled satellites as compared with the
integral of the entire 13C signal of the respective carbon
atom.
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Fig. 6.
13C- Labeling patterns of
verrucosan-2 -ol. 13C-13C coupling of
high signal intensity via single bonds is shown by bold
lines. A, from [1-13C]acetate, highly
enriched carbon atoms are shown by ; B, from
[2-13C]acetate, highly enriched carbon atoms are shown by
; C, from [1,2-13C2]acetate,
pairs of incorporated 13C atoms are connected by bold
lines; D, dissection of isoprenoid monomers on the
basis of the cyclization mechanism proposed in Fig. 10; carbon atoms
contributed by individual monomers are boxed; for details
see "Discussion."
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The labeling patterns of verrucosan-2
-ol samples obtained from
growth with [1-13C]- and [2-13C]acetate
followed a reciprocal pattern (Fig. 6). Eight of the 20 carbon atoms
were predominantly labeled from the carboxylic group of acetate, and
the other 12 carbon atoms were predominantly labeled from the methyl
group of acetate. A statistical analysis is shown in Table III. The 12 atoms which become highly enriched from [2-13C]acetate
have an average enrichment of 62 ± 4%, and the 8 unlabeled atoms
in this sample have an average enrichment of 3 ± 0.1%.
The expected diversion of precursor atoms to isoprenoid monomers via
the mevalonate pathway and via the deoxyxylulose pathway is summarized
in Fig. 7. The mevalonate pathway
implicates the contribution of 3 methyl carbon atoms and 2 carboxyl
carbon atoms from acetate. The deoxyxylulose pathway implicates the
incorporation of all 3 carbon atoms of the putative triose phosphate
precursor and both carbon atoms of activated acetaldehyde, which is
derived from pyruvate by decarboxylation (23, 24, 27). This pathway involves a skeletal rearrangement of 1-deoxyxylulose 5-phosphate conducive to disruption the contiguity of the 3 atoms derived from the
triose precursor.

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Fig. 7.
Diversion of carbon atoms from precursors to
isoprenoid monomers by the mevalonate pathway and the deoxyxylulose
pathway based on data from previously published reports (23-33).
The deoxyxylulose pathway involves a rearrangement of 1-deoxyxylulose
5-phosphate which interrupts the contiguity of the carbon atoms derived
from the putative triose phosphate precursor.
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The reciprocal labeling patterns of verrucosan-2
-ol described above
are in line with the mevalonate pathway which predicts the diversion of
label from the methyl group to 12 carbon atoms of verrucosan-2
-ol
and from the carboxylic group of acetate to the other 8 carbon atoms. A
detailed analysis which confirms this initial working hypothesis is
presented in the Discussion.
In all incorporation experiments, some of the 13C signals
of verrucosan-2
-ol show intense 13C-13C
coupling satellites indicating the presence of two or more
13C atoms in adjacent positions (Fig. 5). The location of
the 13C atom pairs was obtained from two-dimensional
INADEQUATE spectra (Fig. 8). Groups of
adjacent 13C atoms are marked in Fig. 6, A-C, by
bold lines connecting the respective carbon atoms.

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Fig. 8.
Part of a two-dimensional INADEQUATE spectrum
of verrucosan-2 -ol from growth experiment with
[1,2-13C2]acetate. The
13C-13C coherence observed between C-1 and C-2
lies outside the spectral range shown.
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The occurrence of adjacent pairs of 13C atoms is not
surprising in the experiment with double-labeled
[1,2-13C2]acetate, since the mevalonate
pathway is conducive to the incorporation of intact acetate units in
each isoprenoid monomer. Eight pairs of 13C atoms would
thus be expected for a diterpene, of which seven were observed
experimentally (Figs. 6C and 8).
However, adjacent pairs of 13C atoms were also observed in
the experiments with single-labeled acetates. Three contiguous pairs of
13C atoms (>60% satellite intensity relative to overall
13C signal intensity) are present in the biosynthetic
verrucosan-2
-ol from [1-13C]acetate (Fig.
6A). One pair and a triplet of 13C atoms are
present in the compound from the experiment with
[2-13C]acetate (Fig. 6B).
 |
DISCUSSION |
The generation of a tetracyclic ring system from a linear
precursor requires the formation of 4 carbon carbon bonds. If the linear precursor were biosynthesized from acetate via the mevalonate pathway, each carbon atom derived from the methyl group of acetate should be flanked exclusively by carbon atoms derived from the carboxylic group and vice versa (Fig.
9). Thus, each 13C-enriched
carbon would be exclusively bonded to nonenriched neighbor atoms in
experiments with [1-13C]- and
[2-13C]acetate. Only the feeding of
[1,2-13C2]acetate would be conducive to a
linear precursor with directly adjacent 13C atoms
by incorporation of the double-labeled acetate unit. This suggests that
the pairs of directly adjacent 13C atoms observed in the
terpene biosynthesized from single-labeled acetate (i.e.
[1-13C]- and [2-13C]acetate) are generated
by the cyclization reaction. More specifically, the three pairs of
adjacent 13C atoms in the experiment with
[1-13C]acetate signal the formation of 3 carbon carbon
bonds by the cyclization process (Fig. 6A). The formation of three
additional bonds by the cyclization is indicated by the
13C-13C coupling data in the experiment with
[2-13C]acetate (Fig. 6B). It follows that a
minimum of 6 carbon carbon bonds were formed de novo during
the cyclization of the linear precursor. Since the formation of a
tetracyclic system from a linear precursor requires only four
additional bonds, it follows further that a minimum of two carbon
carbon bonds must have been broken during the cyclization. Direct
evidence is available for the breaking of one carbon carbon bond since
the number of 13C-labeled carbon pairs as determined by the
experiment with [1,2-13C2]acetate is 7, whereas 8 intact acetate units should have been present in the linear
C20 precursor (Fig. 9). Thus the reaction mechanism must
imply the breaking of a second carbon carbon bond, which does not
result in the separation of two carbon atoms transferred jointly from a
given acetate molecule.

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Fig. 9.
Labeling pattern of linear C20
terpene precursors predicted by the mevalonate
pathway. Symbols are the same as in Fig. 6. The conformation of
geranyllinaloyl pyrophosphate 8 was selected to match the
structure of the verrucosane diterpene.
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A possible biosynthetic scheme which fulfills all of these criteria is
shown in Fig. 10. Solvolysis of the
allylic pyrophosphate group of (S)-geranyllinaloyl
pyrophosphate (compound 8), triggers off a cyclization
process leading via the monocyclic intermediate 9 to the
bicyclic ionic intermediate 10. The
(S)-configuration of the precursor can be predicted from the configuration of the chiral center in intermediate 9 and the
known anti-stereochemistry of similar SN2'
bond-making processes (37, 38). Next, ion 10 suffers a
1,2-rearrangement leading to intermediate 11, the cationic
center of which then attacks the isopropylidene double bond with
formation of the tricyclic ion 12. Saturation of the
positive charge in this ion is best accommodated by a 1,5-hydride
transfer from the C-2 methylene group which generates the homoallylic
intermediate 13. Similar shifts have been observed
previously in terpene biosynthesis (39, 40). Collapse of intermediate
13 to the cyclopropylcarbinyl ion 14 is followed
by a sigmatropic rearrangement to a new cyclopropylcarbinyl ion
15 and the reaction is terminated by addition of a hydroxyl
group from the solvent. The interconversion of the two
cyclopropylcarbinyl ions 14 and 15, which may but
need not require the intermediacy of a cyclobutyl cation, is well
precedented in abiotic and biological systems (41).
The mechanism outlined in Fig. 10 complies with the minimum
requirements for bond-making and bond-breaking processes specified by the experimental data, namely the formation of six carbon bonds and
the disruption of two carbon bonds. In addition, it provides a
rationale for the stereochemical course of the events which lead to the
formation of the isopropyl side chain of verrucosan-2
-ol. The trans
arrangement of the two hydrogen atoms at C-13 and C-14 in the final
product of the cyclization requires a boat conformation for the chain
segment of the aliphatic precursor which is being used for the
formation of the pentacyclic ring (Fig.
11). Inspection of models indicates
that in the ionic intermediate 12 issued from this
cyclization step one of the lobes of the empty p-orbital is
ideally positioned for abstracting a hydride ion from the
-position of the C-2 methylene group; as a consequence, the postulated
1,5-hydride shift predicts a correlation between the
(Z)-methyl group of the aliphatic precursor (dotted
line in Fig. 11) and the Re-methyl group in the
isopropyl chain of the final compound. This prediction is born out by
the results of the feeding experiment with
[1,2-13C2]acetate, which show that within the
isopropyl chain of verrucosan-2
-ol it is the Re-methyl
group (responsible for signals at 15.08 ppm in the 13C NMR
and at 0.80 ppm in the 1H NMR spectra of the compound) that
maintains its bond-labeling to the adjacent atom. Therefore, this group
must have been generated specifically from the (Z)-methyl
group in the isopropylidene unit of the aliphatic precursor, which is
known to correspond to the methyl group of mevalonate (42-44).
On the basis of the proposed cyclization mechanism, the four isoprenoid
monomers in verrucosan-2
-ol can now be identified unequivocally. The
carbon atoms arising from each monomer are boxed in Fig.
6D. Based on this dissection, it is possible to extract
unequivocally the labeling patterns of the isoprenoid monomers from the
labeling data of verrucosan-2
-ol shown in Fig. 6, A-C,
and in Table IV. Moreover, the 13C enrichments and also the
relative contribution of the coupling satellites can be averaged over
all four monomers. The averaged data and their standard deviations are
summarized in Table V. The essential
features are also shown graphically in the central column of Fig.
12.

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Fig. 12.
Observed and predicted labeling patterns for
isoprenoid monomers. For details, see Fig. 4 and
"Discussion."
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The hypothetical labeling patterns of isoprenoids via the mevalonate
pathway and via the deoxyxylulose pathway can be predicted on basis of
the experimentally determined labeling patterns of acetate, pyruvate,
and glyceraldehyde 3-phosphate. The labeling patterns predicted for the
mevalonate pathway on basis of the acetyl-CoA pattern are shown in the
left column of Fig. 12. The labeling patterns predicted for
the deoxyxylulose pathway on basis of the pyruvate and glyceraldehyde
3-phosphate-labeling patterns are shown in the right column,
and the experimental data are shown in the center.
The labeling patterns predicted by the mevalonate pathway agree well
with the experimental observation. On the other hand, the patterns
predicted by the deoxyxylulose pathway do not agree with the
experimental data. Specifically, the deoxyxylulose pathway predicts
high enrichment of C-3 of IPP formed from [2-13C]acetate
in contrast to the virtual absence of label (3%) observed experimentally. It also predicts extensive
13C-13C coupling in the experiment with
[2-13C]acetate which is not observed.
On basis of the data in Fig. 12, the isotope distribution predicted by
the two different pathways can be projected to the entire verrucosan-2
-ol molecule. The labeling predictions for both pathways and the experimental data for the incorporation of
[2-13C]acetate are shown in Fig.
13. The agreement between the
experimental data and the mevalonate prediction based on the labeling
of intracellular acetyl-CoA is very close. It should be noted that not
all observed couplings can be unequivocally and quantitatively assigned
to individual pairs of carbon atoms because the coupling constants cluster in a narrow range, and it is not always possible to determine the contributions of individual pairs in the complex isotopomer mixture. Only couplings which can be assigned unequivocally are shown
for the structure in the center of Fig. 13.

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Fig. 13.
Observed and predicted labeling patterns for
verrucosan-2 -ol after feeding with
[2-13C]acetate. For details, see Fig. 4 and
"Discussion."
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Using the data of Fig. 13, the 13C NMR signals of
verrucosan-2
-ol were simulated. The data (not shown) demonstrate
again that only the mevalonate prediction agrees with the experimental
data.
Knowledge on the distribution of the two isoprenoid pathways in
different species is still limited. The eubacteria E. coli and Z. mobilis have been shown to use the deoxyxylulose
pathway (26, 27), but the primitive eubacterium C. aurantiacus uses the mevalonate pathway which is also
characteristic of animal metabolism. The deoxyxylulose pathway has been
shown to be involved in the biosynthesis of several plant metabolites
such as gingkolides in Gingko biloba (23), taxoids in the
yew (29), loganin in Rauwolfia serpentina (31), plastidic
isoprenoids of barley, duckweed, and carrot (32), the prenyl side
chains of several metabolites in the green alga Scenedesmus
obliquus (28), and monoterpenoids in Mentha piperita
(33). In their studies with G. biloba, Arigoni and his
co-workers (23, 24) showed that steroids are formed in the cytoplasm
via the mevalonate pathway and gingkolides in plastids via the
deoxyxylulose pathway. The presence of different pathways in the
cytoplasm and in organelles could be related to the endosymbiontic
origin of organelles, but the distribution of the two pathways in the
microbial kingdom is as yet insufficiently known to serve as a
theoretical basis for evolution arguments. Horizontal gene transfer
between distant species may also have to be considered.
We thank Gerhard Heßler for help with the
molecular dynamics calculation and Angelika Werner for expert help with
preparation of the manuscript.