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The discovery of a mevalonate-independent pathway for
isoprenoid biosynthesis in bacteria, algae and higher plants†
Michel Rohmer
Université Louis Pasteur - CNRS, Institut Le Bel, 4 rue Blaise Pascal, 67070 Strasbourg Cedex,
France. E-mail: [email protected]
Received (in Cambridge) 23rd March 1999
Covering: up to end of December 1998
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1
1.1
1.2
2
2.1
2.2
2.3
3
3.1
3.2
4
5
5.1
5.2
5.3
6
7
8
1
1.1
Isoprenoid biosynthesis
The mevalonate route to isopentenyl diphosphate
Isoprenoid biosynthesis in higher plants: some
contradictions with the mevalonate pathway
The discovery of the mevalonate-independent
pathway
The origin of the discovery: the biosynthesis of
bacterial hopanoids
The origin of the carbon atoms of isoprenic units in
the mevalonate-independent pathway
d-Glyceraldehyde 3-phosphate and pyruvate as the
first precursors of isopentenyl diphosphate
Towards the identification of intermediates and
enzymes of the new pathway
1-Deoxy-d-xylulose 5-phosphate and
1-deoxy-d-xylulose 5-phosphate synthase
2-C-Methyl-d-erythritol 4-phosphate and
1-deoxy-d-xylulose 5-phosphate reducto-isomerase
The distribution of the glyceraldehyde
3-phosphate/pyruvate pathway amongst prokaryotes
The distribution of the GAP/pyruvate pathway
amongst phototrophic eukaryotes
Essential plant chloroplast isoprenoids and sterols
from green algae
Isoprenoids from secondary metabolism
Intermediate exchanges between the mevalonate and
the GAP/pyruvate pathways in plants
Conclusion
Acknowledgments
References
Isoprenoid biosynthesis
The mevalonate route to isopentenyl diphosphate
Isoprenoids are present in all living organisms. Formally, they
are derived from the branched C5 carbon skeleton of isoprene,
and the number of repetitions of this motif, cyclization
reactions, rearrangements and further oxidation of the carbon
skeleton are responsible for the enormous diversity of structures.1 Isoprenoids include essential metabolites. Sterols (18)
(Fig. 1), which are amongst the most studied, are present in most
eukaryotes, acting as membrane stabilizers, and in verterbrates
as precursors for steroid hormones and bile acids. Carotenoids
(15) (Fig. 1) are essential constituents of the photosynthetic
apparatus in all phototrophic organisms. Long acyclic isoprenic
chains are found in the quinones of electron transport chains,
such as ubiquinone 12, menaquinone 13 or plastoquinone 14 or
† Abbreviations used: DMAPP: dimethylallyl diphosphate; DX: 1-deoxy-dxylulose; DXP: 1-deoxy-d-xylulose 5-phosphate; FPP: farnesyl diphosphate; GAP: d-glyceraldehyde 3-phosphate; GGPP: geranylgeranyl
diphosphate; GPP: geranyl diphosphate; HMGCoA: 3-hydroxy-3-methylglutaryl coenzyme A; IPP: isopentenyl diphosphate; ME: 2-C-methyl-derythritol; MEP: 2-C-methyl-d-erythritol 4-phosphate; MVA: mevalonic
acid.
OH
OH
OH
NH2
11
O
O
CH3O
CH3O
H
H
n
O
n
O
12
13
OH
14
O
H
9
O
16
15
R
O
O
O
O
O
OH
HO
O
O
HO
17
18
Fig. 1 Examples of investigated isoprenoids for the elucidation of the
GAP/pyruvate pathway. Bacterial isoprenoids: aminobacteriohopanetriol
(hopanoid) 11, ubiquinone 12, menaquinone 13. Chloroplast isoprenoids:
phytol 14, b-carotene 15, plastoquinone 16. Plant diterpenoid: ginkgolide A
17. Sterols 18.
in polyprenols 10, such as the dolichols from eukaryotes and the
bactoprenol from eubacteria, serving as carbohydrate carriers.
The most numerous isoprenoids however may be considered as
secondary metabolites of less obvious role: for instance the
mono-, sesqui- and diterpenes from plant essential oils and
resins.
Feeding experiments with isotopically labeled precursors
were performed very early in the field of isoprenoid biosynthesis. The first incorporations concerned the biosynthesis
of cholesterol in liver tissues and ergosterol in yeast. This
revealed a biosynthetic pathway (Scheme 1), starting from
acetate, activated as acetyl coenzyme A 1, and yielding IPP 5
which is the biological equivalent of isoprene and presents the
basic branched C5 skeleton of the isoprenic unit.2,3 Prenyltransferases catalyze the condensation of IPP 5 on to
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O
■
O
•
+
•
■
O
O
•
•
■
SCoA
1
■
SCoA
■
■
HO
■
•
O
•
HO
■
•
OH
SCoA
2
•
O
O
4
O
SCoA
O
■
■
2 NADPH
•
■
•
SCoA
O
3
3 ATP
5
■
•
■
1
3
■
4
•
■
2
OPP
■
•
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5
Scheme 1
•
■
OPP
6
Mevalonate pathway for isoprenoid biosynthesis.
DMAPP 6 or on to other prenyl diphosphates (7 to 10) to give
the higher homologues which are the precursors of all
isoprenoid series (Scheme 2). The committed step of this
C5
+
OPP
OPP
6
5
topically labeled MVA and acetate were usually not, or were
only very poorly, incorporated into carotenoids, monoterpenes
and diterpenes in plant systems.4–9 In contrast, these precursors
were always efficiently incorporated into the sterols, the
triterpenoids and quite often also into the sesquiterpenoids.
Furthermore, mevinolin, a potent inhibitor of HMGCoA
reductase, the enzyme catalyzing the committed step of the
MVA pathway, strongly inhibited sterol biosynthesis in plants,
but did not affect the formation of the chloroplast pigments such
as the carotenoids and the chlorophylls containing the diterpenic
phytyl side-chain.10,11 Such results were in most cases interpreted in terms of lack of permeability of the chloroplast
membrane towards the precursor or the inhibitor. In addition,
neither MVA, nor MVA phosphate or MVA diphosphate were
incorporated into isoprenoids by a carefully purified fraction of
spinach chloroplasts or daffodil chromoplasts, and the key
enzymes of the MVA pathway could not be characterized in
these plant systems.12,13 IPP was however well incorporated,
pointing out the unquestionable role of this compound as
isoprenoid precursor in the chloroplasts. An independent IPP
biosynthesis via the MVA pathway was consequently postulated in the chloroplasts, although the possible presence of
another route was not excluded.14 As a matter of fact, the second
metabolic route towards the isoprenoids was first detected in
bacteria and later found to be widespread amongst phototrophic
eukaryotes.
IPP
2
C10
monoterpenes
OPP
7
IPP
sesquiterpenes
triterpenes
C15
OPP
8
IPP
C20
OPP
9
C5n
OPP
n
diterpenes
carotenoids
polyprenols
ubiquinones
menaquinones
plastoquinones
10
Scheme 2 Biosynthesis of higher isoprenoids from isopentenyl diphosphate 5 and dimethyallyl diphosphate 6: geranyl diphosphate 7, farnesyl
diphosphate 8, geranylgeranyl diphosphate 9, polyprenyl diphosphates
10.
metabolic route is the reduction of HMGCoA 3 by a reductase,
yielding MVA 4. This biosynthetic scheme (Scheme 1) was
universally accepted for the biosynthesis of all isoprenoids in all
living organisms despite some contradictory results essentially
obtained in the field of the isoprenoid biosynthesis in plants.
1.2 Isoprenoid biosynthesis in higher plants: some
contradictions with the mevalonate pathway
As early as the 1950’s, contradictions with the universal role of
MVA as isoprenoid precursor were made apparent and
subsequently became even more obvious. In particular, iso566
Nat. Prod. Rep., 1999, 16, 565–574
The discovery of the mevalonate-independent pathway
2.1 The origin of the discovery: the biosynthesis of
bacterial hopanoids
Except for two species, bacteria do not synthesize sterols. This
is in sharp contrast with the omnipresence of sterols in most
eukaryotes. Only triterpenoids of the hopane series 11 (Fig. 1)
are found, scattered in a fair number of eubacteria.15,16 The
structural similarities between bacterial hopanoids and eukaryotic sterols suggested similar functional roles. Indeed, in
membrane models hopanoids act, at least qualitatively, as
membrane stabilizers, much like sterols do.17 The presence of
hopanoids was correlated with the tolerance towards high
temperature of the thermoacidophilic Alicyclobacillus acidocaldarius,18 towards ethanol and high osmotic pressures of
Zymomonas mobilis19 or protection of the nitrogenase from
oxygen in the nitrogen fixing bacteria Frankia sp. and
Bradyrhizobium japonicum.20,21 Hopanoids are usually present
in concentrations of the same order of magnitude as those found
for sterols in eukaryotic cells.15 Such concentrations are in most
cases at least one order of magnitude higher than those of the
ubiquitous bacterial isoprenoids such as the ubiquinones 12 and
the menaquinones 13 or of the bactoprenol, and even higher
than those of most bacterial carotenoids. Hopanoids are
chemically stable and easily isolated. This makes them well
suited for NMR spectroscopy and therefore for labeling
experiments using stable isotopes. Although the early steps of
isoprenoid biosynthesis were only poorly investigated in
bacteria, no surprise was expected for the formation of their
isoprenic units. Our first labeling experiment with hopanoid
producing bacteria was justified by a unique feature of these
natural products: an additional C5 non-isoprenic side-chain of
unknown origin is linked by a carbon–carbon bond to the
triterpenic skeleton.22 The experiments were performed with
bacteria capable of growing on a culture medium of definite
composition using a single carbon source. Such conditions
should facilitate the interpretation of the observed labeling
patterns as only a few and, hopefully, well identified metabolic
routes were involved in the metabolism of a single 13C labeled
carbon source. Even in the case of the phototrophic bacterium
Rhodopseudomonas palustris, heterotrophic growth conditions
in the dark were chosen. This avoided a possible uniform
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labeling by recycling of 13CO2 issued from the catabolism of the
labeled carbon source. These labeling conditions distinguished
our incubations from most previous experiments performed
with bacteria, using complex culture media of undetermined
composition and only leading to partly interpretable results,
probably because of the competition for the utilization of
numerous carbon sources and the intervention of several
metabolic pathways.22–25
The first labeling experiments were performed using [1-13C]and [2-13C]acetate with two heterotrophically grown Rhodopseudomonas species (R. palustris and R. acidophila) and with
Methylobacterium organophilum.22 They allowed us to determine the origin of the bacteriohopane side-chain from a dpentose, which is synthesized via non-oxidative pentose
phosphate metabolism and which is linked via its C-5 carbon
atom to the isoprenyl group of the triterpenic moiety. The
labeling pattern in the isoprenic units was expected to be trivial
and simply to result from the MVA route. It was however
completely different and did not result from the direct
incorporation of acetate via the classical MVA pathway.
Labeling patterns, especially those obtained from [1-13C]acetate, were clear (Scheme 3). No scrambling was observed.
Acetate was utilized by bacteria as the single carbon source after
incorporation into the glyoxylate and tricarboxylic acid cycles
and was consequently incorporated into IPP via further
metabolic routes that had to be identified. In a first attempt,
these results were interpreted in the frame of the MVA route
which had no reason to be rejected at that time, assuming the
compartmentation of acetate metabolism and the existence of
two different and non-interconvertible activated acetate pools,
although a completely different route could not be excluded.22
Furthermore, incorporation of 13C labeled acetate was later
performed with Escherichia coli, a bacterium producing no
hopanoids: again the isoprenic units of the ubiquinone prenyl
chain presented the same labeling pattern from acetate as those
previously found in the hopanoids from the former bacteria,
indicating a probable general distribution of this metabolic
pathway.
2.2 The origin of the carbon atoms of isoprenic units in
the mevalonate-independent pathway
Useful clues to the origin of the carbon atoms of isoprenic units
in bacteria and for the elucidation of the MVA independent
route (Scheme 4) were obtained from labeling experiments
using 13C labeled glucose isotopomers with Zymomonas
mobilis, a facultative anaerobic and fermentative bacterium.26
Z. mobilis utilizes only hexoses, essentially glucose, as a carbon
1. Glyoxylate and tricarboxylic acid cycles
•CO2H
O
O
• 2H
CO
CH3
•
•
CO2H
A
CH3
CH3
CO2H
O
B
OP
CO2H
•
HO
•
•CHO
CH2
•
OH
OH
OPP
CH2OP
CH2OP
2. Entner-Doudoroff pathway
CO2H
CHO
O
CO2H
CH3
•CH3
O
OH
OH
OH
OH
OH
•CH2OH
•CH2OP
•
O
CH2
HO
B
HO
•
•
OPP
OH
CHO
CH2OP
OH
•CH2OP
3. Glycolysis
CH2OP
CHO
O
OH
CH2OH
HO
HO
OH
OH
•
•
CH2OP
CO2H
OH
CH2OH
OH
OH
CH2OH
CHO
O
•CH3
O
•CH2OP
•CH3
B
O
O
•CH3
HO
SCoA
OH
•
CH2OP
C
•
•
•
OPP
•
•
OPP
Scheme 3 Incorporation of acetate and glucose into isoprenoids:(A): glyoxylate and tricarboxylic acid cycles.(B): GAP/pyruvate pathway.(C): MVA
pathway. 1. Acetate metabolism via the glyoxylate and tricarboxylic acid cycles and incorporation of the resulting GAP and pyruvate into isoprenoids via
the non-MVA route (e.g. in those of Escherichia coli and Rhodopseudomonas palustris). 2. Glucose catabolism via the Entner–Doudoroff pathway and
incorporation of GAP and pyruvate via the non-MVA pathway in the isoprenoids of e.g. Methylobacterium fujisawaense. 3. Glucose catabolism via glycolysis
and incorporation of GAP and pyruvate (B) via the non-MVA pathway (e.g. in the isoprenoids of E. coli or of chloroplasts) or (C) via the acetyl-CoA/MVA
route (e.g. in plant sterols).
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O
N
O
N
CO2
OP
H
S
S
19
O
H
OH
OH
21
20
H
H
N
O
OH
O
OP
OP
S
OH
OH
22
HO
OP
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O
OH
HO
OH
23
NADPH
OH
PO3H2
N
OP
O
OH
24
25
OPP
5
Scheme 4 Mevalonate-independent glyceraldehyde 3-phosphate/pyruvate pathway for isoprenoid biosynthesis.
source. Glucose is metabolized via the Entner–Doudoroff
pathway (Scheme 3) and is efficiently converted under
anaerobic growth conditions into ethanol. Glucose isotopomers
with labeling at either C-1, C-2, C-3, C-5 or C-6 were added to
the culture medium of Z. mobilis. The resulting labeling pattern
found in the hopanoids allowed us to determine the origin of the
carbon atoms in the isoprenic units. Carbon atoms corresponding to C-3 and C-5 of IPP had a dual origin: they were
respectively equally derived from C-2 or C-5 and C-3 or C-6 of
glucose. Those derived from C-1, C-2 and C-4 of IPP were
solely derived from C-4, C-5 and C-6 of glucose respectively.24
Such a labeling pattern was in accordance with pyruvate 19 as
precursor of a C2 subunit and a triose phosphate derivative 21 as
precursor of a C3 subunit (Scheme 4). Indeed, in Z. mobilis,
pyruvate is equally obtained via the Entner–Doudoroff pathway
either directly from the ‘upper’ part of glucose (C-1 to C-3) or
from the ‘lower’ part (C-4 to C-6) via GAP yielding after
mixing of these two non-discernible pools and after decarboxylation a C2 unit equally derived from C-2/C-3 and C-5/C-6 of
glucose. Most interestingly, pyruvate is not converted by Z.
mobilis into GAP.26 Thus, a triose phosphate derivative is solely
derived in this bacterium from the three carbon atoms C-4, C-5
and C-6 of glucose and therefore represents a reasonable
precursor for a C3 subunit in the isoprenoid biosynthesis. Such
an interpretation was confirmed by 13C glucose feeding
experiments performed with Methylobacterium fujisawaense
(Scheme 3).24 This bacterium also utilizes glucose via the
Entner–Doudoroff pathway, but presents the normal sequence
of enzymatic reactions allowing the interconversion of pyruvate
and GAP. Labeling patterns observed in the isoprenic units of
the hopanoids from this bacterium resembled those previously
found for Zymomonas mobilis. The only difference was that the
C3 subunit was equally derived from C-1, C-2 and C-3 or C-4,
C-5 and C-6.
Another confirmation was obtained with a bacterium utilizing glucose via glycolysis (Scheme 3). Incorporation of [1-13C]and [6-13C]glucose into ubiquinone in E. coli or into the
hopanoids in Alicyclobacillus acidoterrestris yielded identical
labeling patterns with label on all carbon atoms corresponding
to C-1 and C-5 of IPP.24 Cleavage of fructose 1,6-biphosphate
by the aldolase yielded GAP and dihydroxyacetone phosphate
which are interconverted by triose phosphate isomerase. This
leads to the equivalence of the resulting two moieties of glucose
(C-1 to C-3 and C-4 to C-6). All carbon atoms derived from C-1
568
Nat. Prod. Rep., 1999, 16, 565–574
of IPP (arising from C-3 of a triose phosphate derivative) and
from C-5 of IPP (arising from C-3 of pyruvate) were
indifferently labeled from [1-13C]- or [6-13C]glucose.
The labeling patterns formerly observed after the first feeding
experiments of 13C labeled acetate were now clear. They
resulted from the insertion of acetate into the glyoxylate cycle
and the tricarboxylic acid cycle, yielding phosphoenolpyruvate
from oxaloacetate and consequently pyruvate and the triose
phosphate derivative which were incorporated into the isoprenic
units (Scheme 3).22,24
These labeling patterns found in bacterial isoprenoids were
clearly not compatible with the MVA pathway (Scheme 3) and
MVA could be excluded as an intermediate in the novel
bacterial metabolic route to IPP. On the one hand, neither MVA,
nor MVA phosphate or MVA diphosphate, were converted into
IPP by cell-free systems from E. coli and Z. mobilis, two
bacteria showing the odd labeling patterns after feeding of 13C
labeled precursors.27 They were however efficiently incorporated into IPP by cell free systems from prokaryotes possessing,
according to the literature, the MVA route such as Staphylococcus carnosus, Myxococcus fulvus or Halobacterium cutirubrum.27 In parallel, HMGCoA reductase, the key enzyme of the
MVA pathway, could not be detected in E. coli and Z. mobilis,
whereas it was definitely present in the second group of
microorganisms.27
Clearly, if a triose phosphate derivative is a precursor for the
carbon atoms C-1, C-2 and C-4 of IPP, this implies that a
rearrangement is involved in the formation of the branched
isoprenic skeleton allowing the insertion of the C2 subunit
derived from pyruvate decarboxylation between two carbon
atoms of the C3 subunit (Scheme 4). Incorporation of doubly
labeled [4,5-13C2]glucose into the hopanoids and ubiquinone of
Methylobacterium fujisawaense shed for the first time light on
such an intramolecular rearrangement. In accord with glucose
catabolism via the Entner–Doudoroff pathway, all carbon atoms
corresponding to C-2 and C-4 of IPP were labeled. Owing to the
sufficiently high isotopic abundance in the enriched positions,
their signals appeared as doublets with characteristic 2J 13C–13C
coupling constants.24 The conservation of this coupling indicated that the C-4 and C-5 carbon atoms of glucose are
simultaneously introduced into isoprenic units from the same
glucose molecule, and represent the signature of an intramolecular transposition for the formation of the IPP carbon
skeleton. Isoprenic units in the hopanoids for Z. mobilis were
also derived from a C2 and a C3 subunit as shown by
incorporation of uniformly labeled [U-13C6]glucose. On the one
hand, a 1J 13C–13C coupling was observed between all carbon
atoms derived from C-3 and C-5 of IPP: this was also a
signature that the C2 subunit derived from C-2 and C-3 of
pyruvate, after loss of C-1 by decarboxylation. On the other
hand, 1J 13C–13C coupling constants between carbon atoms
derived from C-1 and C-2 of IPP were accompanied in some
isoprenic units by long range couplings 2J and 3J characteristic
of the intervention of the C3 subunit and the rearrangement
reaction formerly discovered by using the doubly labeled
[4,5-13C2]glucose.28
The same labeling patterns as those found in the bacteria
utilizing glucose via the Embden–Meyerhof–Parnas route were
independently found for the formation of the diterpenoids from
higher plants.29 After feeding Ginkgo biloba embryos with
[1-13C]- or [6-13C]glucose, the distribution of the isotope in
ginkgolides 17 (Fig. 1) and bilobalides was identical with that
observed for the isoprenoids of E. coli or Alicyclobacillus
acidoterrestris. Additional experiments using [2-13C]- and
[3-13C]glucose as well as uniformly labeled [U-13C6]glucose
confirmed the formation of IPP from pyruvate and a triose
phosphate intermediate. This represented the first proof for the
presence of the MVA-independent route in a plant and indicated
that its occurrence was not restricted to eubacteria. In the
following sections, more data on the widespread distribution of
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this metabolic route in phototrophic eukaryotes will be
presented.
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2.3 d-Glyceraldehyde 3-phosphate and pyruvate as the
first precursors of isopentenyl diphosphate
The direct involvement of pyruvate 19 as the source of the C2
subunit (Scheme 4) was obtained by labeling of the hopanoids
and the ubiquinone of Methylobacterium fujisawaense using
[3-13C]pyruvate in the presence of acetate or glucose.24 Only
carbon atoms derived from C-5 of IPP were labeled. Such an
outcome had previously been observed after the incorporation
of [3,3,3-2H3]lactate into ubiquinone in E. coli, but the
observation could not be interpreted in terms of a completely
novel pathway for isoprenoid biosynthesis.30 Confirmation was
also obtained in the case of the prenyl chains of ubiquinone and
menaquinone in E. coli by incorporation of l-[3-13C]lactate in
the presence of glucose: all carbon atoms derived from C-5 of
IPP, and accordingly from the pyruvate moiety, as well as those
derived from C-1 (triose phosphate moiety) were labeled.31 The
latter labeling of the carbon atoms derived from C-1 of IPP
indicated that pyruvate was converted into GAP.
Definitive proof for the identity of the C3 subunit was
obtained using mutants of E. coli which were each defective in
a single enzyme of the triose phosphate metabolism and were
accordingly unable to interconvert pyruvate and glycerol.28
These mutants were grown on a defined medium containing
simultaneously only pyruvate and glycerol as carbon sources.
Two types of labeling experiments were performed with each
mutant: the first one with 13C labeled pyruvate and unlabeled
glycerol, and the second one with unlabeled pyruvate and 13C
labeled glycerol. The resulting labeling patterns were determined in the prenyl chain of ubiquinone. All mutants incorporated label from pyruvate into the C2 subunit, clearly confirming
that it arose from pyruvate decarboxylation. In the C3 subunit ,
in contrast, labeling from pyruvate was only found in those
mutants lacking glycerol kinase, glycerol phosphate dehydrogenase and triose phosphate isomerase, whereas labeling
from glycerol was only detected in those lacking enolase,
phosphoglycerate isomerase and glyceraldehyde phosphate
dehydrogenase. These results were only compatible with a key
role for GAP 21 as the C3 moiety involved in the formation of
isoprenic units (Scheme 4).
3 Towards the identification of intermediates and
enzymes of the new pathway
3.1 1-Deoxy-d-xylulose 5-phosphate and
1-deoxy-d-xylulose 5-phosphate synthase
Cell-free systems or enzymic preparations from numerous
bacteria, fungi and yeasts were capable of synthesizing
1-deoxy-d-xylulose (1-deoxy-d-threo-pentulose) or its 5-phosphate 22 from pyruvate and from d-glyceraldehyde or from
GAP (Scheme 4).32,33 The enzymic activity was thiamine
diphosphate dependent and probably related to that of pyruvate
dehydrogenase. The reaction was not specific: the system
accepted acyloins in place of pyruvate as acetyl donor and
different aldoses, yielding 1-deoxyketoses with C5, C6 or C7
skeletons. The 1-deoxy-d-xylulose C5 chain was also shown to
be the precursor for the heterocycles of thiamine34,35 and
pyridoxol.36 Its role in isoprenoid biosynthesis was first
recognized in E. coli.31 Indeed, [1-2H]- and [5,5-2H2]deoxyxylulose were efficiently incorporated into the prenyl side-chain
of ubiquinone and menaquinone in E. coli. The labeling found
on the carbon atoms respectively derived from C-5 and C-1 of
IPP was as expected from the MVA-independent pathway.
Incorporation of doubly labeled [2,3-13C2]- and [2,4-13C2]deoxyxylulose into the ubiquinone in the bacterium E. coli37
and of multiply labeled [2,3,4,5-13C4]deoxyxylulose into phytol
and carotenoids of cell cultures of the higher plant Catharanthus roseus38 confirmed the formation of the branched
isoprenic skeleton by an intramolecular rearrangement. These
experiments also showed that the pentulose was incorporated
intact into the isoprenic units, without previous degradation.
Accordingly, the five carbon atoms of IPP exactly correspond to
those of deoxyxylulose.
The gene of the deoxyxylulose 5-phosphate synthase was
identified owing to its homology with those of transketolases,
other thiamine diphosphate dependent enzymes. The gene was
cloned in the bacterium E. coli39,40 and in a higher plant,
Mentha 3 piperita,41 and overexpressed in E. coli. The enzyme
catalyzes the concomitant decarboxylation of pyruvate 19 and
the condensation of the resulting (hydroxyethyl)thiamine 20 on
free d-glyceraldehyde or GAP 21, respectively yielding deoxyxylulose or deoxyxylulose 5-phosphate 22 (Scheme 4). As
free glyceraldehyde is not a usual cellular metabolite, GAP and
DXP are probably the normal substrate and reaction product
formed in vivo by the synthase.
3.2 2-C-Methyl-d-erythritol 4-phosphate and
1-deoxy-d-xylulose 5-phosphate reducto-isomerase
A rearrangement is involved in the formation of the isoprenoid
skeleton by the mevalonate-independent route. From the
beginning, the equivalent of an acid catalyzed a-cetol rearrangement was postulated (Scheme 4).24 A similar transposition, catalyzed by the acetolactate reducto-isomerase, is involved in the formation of the carbon skeleton of a-amino acids
with branched side-chains such as valine. Applied to DX or
DXP, such a rearrangement would yield 2-C-methyl-d-erythrose or its 4-phosphate 23 and after reduction 2-C-methyl-derythritol (ME) or its 4-phosphate (MEP) 24.28 The 2,4-cyclodiphosphate of ME is found in normal growth conditions in the
anaerobic sulfate-reducing bacterium Desulfovibrio desulfuricans 42 and is accumulated under oxidative stress conditions
induced by benzylviologen and other related oxidants by several
aerobic bacteria.43–45 The free tetrol or the 2-C-methyl-derythronolactone were both found in higher plants.46–53 It was
therefore tempting to assess a possible role for ME in the MVA
independent route. Corynebacterium ammoniagenes accumulates large amounts of methylerythritol cyclodiphosphate during the stationary growth phase in the presence of glucose and
benzylviologen. After incubation of this bacterium with
[1-13C]-, [6-13C]- or [U-13C6]glucose, the labeling patterns
found in the isoprenic units of the dihydromenaquinones and in
ME were identical. They corresponded to those expected from
the MVA independent route, indicating that both carbon
skeletons were most probably formed by the same reaction
sequence.54 Furthermore, the deuterium labeled d-enantiomer
of ME was incorporated in low yield, but without ambiguity,
into the prenyl side-chain of ubiquinone by a wild type E. coli
strain whereas the l-enantiomer was not utilized at all.55 This
showed that ME is a possible precursor for isoprenic units. The
low incorporation was probably due to the absence of an
efficient kinase allowing the conversion of the free tetrol into its
4-phosphate, which is probably the required intermediate in this
pathway. Additional data for the formation of ME via the MVA
independent route were obtained for Liriodendron tulipifera.
Feeding senescent leaves of this tree with 13C labeled DX
showed that the accumulated ME derived from this pentulose.56
E. coli mutants which were auxotrophic to ME and requiring
this tetrol for growth were produced. A gene that complemented
in these mutants the region coding for IPP biosynthesis was
cloned. This allowed the identification of the enzyme responsible for the conversion of DXP 22 into MEP 24.57,58 This
reducto-isomerase is NADPH dependent and requires Mn2+, a
co-factor that is less efficiently replaced by Co2+ or Mg2+. It
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catalyzes two consecutive steps: the rearrangement of DXP 22
into 2-C-methyl-d-erythrose 23 and the concomitant reduction
of this aldose into MEP 24 (Fig. 5). The free aldose phosphate
was not detected, and the presence of the 5-phosphate group on
DXP was required for the enzymatic activity. Free DX was not
converted into ME, confirming the former hypothesis that MEP,
rather than ME, is the intermediate involved in isoprenoid
formation. As expected, this reaction closely resembles that
catalyzed by the acetolactate reducto-isomerase.
Fosmidomycin 25, an antibiotic identified several years ago,
presents interesting antibacterial activity against several Gramnegative and some Gram-positive bacteria (Scheme 4).59 It
affects the biosynthesis of the quinones in sensitive bacteria and
has no activity against bacteria that apparently synthesize their
isoprenoid via the MVA pathway. These data suggested that the
target of the antibiotic might be linked to isoprenoid biosynthesis and to the MVA independent route. Fosmidomycin
was recently shown to inhibit efficiently the DXP reductoisomerase of E. coli.60 This antibiotic also interferes with the
biosynthesis of plastidic isoprenoids in higher plants that are
synthesized via the non-MVA pathway (see section 5.1). It
inhibited the de novo biosynthesis of carotenoids and chlorophylls (containing the diterpenic phytol chain) during greening
of etiolated barley seedlings or Lemna gibba plants.61 It also
blocked the emission of isoprene by leaf pieces of Populus
nigra, Platanus 3 acerifolia and Chelidonium majus.61
4 The distribution of the glyceraldehyde
3-phosphate/pyruvate pathway amongst prokaryotes
Even if IPP was usually successfully incorporated into bacterial
isoprenoids, in accordance with its ubiquitous role as isoprenoid
precursor,25 very little information on the early steps of
isoprenoid biosynthesis in these microorganisms is found in the
literature. Attempts at incorporation of 14C labeled acetate or
MVA usually failed or resulted in poor yields. Radioactivity
was in most cases not localized by chemical degradation of the
labeled metabolite. When this was checked, as for instance in
the case of the incorporation of [1-14C]- and [2-14C]acetate in
the ubiquinone of E. coli, the labeling was not found at the
expected positions, clearly indicating that acetate was not
directly incorporated after conversion into acetyl-CoA in the
isoprenic units via the MVA pathway.62 Most of these
anomalous results can be now re-interpreted by the intervention
of the MVA independent route. Since the discovery of this
pathway in the already mentioned prokaryotes that were
selected for its elucidation (Rhodopseudomonas palustris, R.
acidophila, Methylobacterium fujisawaense, M. organophilum,
Escherichia coli, Zymomonas mobilis, Alicyclobacillus acidoterrestris and Corynebacterium ammoniagenes),22,24,54 this
metabolic route has in addition been found in numerous other
bacterial species. Amongst Gram-negative strains, it was
detected in all investigated enterobacteria (Citrobacter freundii,
Escherichia coli, Salmonella typhimurium, Erwinia carotovora), in pseudomonads and related species (Pseudomonas
aeruginosa, P. fluorescens, Burkholderia caryophylli, B. gladioli, Ralstonia pickettii) and in Acinetobacter calcoaceticus63 as
well as in the cyanobacterium Synechocystis sp.64,65 In Grampositive species, the MVA independent route was detected in
the two mycobacteria Mycobacterium phlei and M. smegmatis.63 The MVA independent route is also present in many
Streptomyces species for the formation of sesquiterpenoids
derived from pentalenic acid in Streptomyces sp. UC531966 and
for the formation of the isoprenic moiety of antibiotics such as
carquinostatin B in S. exfoliatus,67 novobiocin in S. niveus68 and
S. spheroides,69 moenomycin A in S. ghanaensis,70 and
teleocidins in S. blastmyceticum.71
More strikingly, Streptomyces aeriouvifer possesses the
GAP/pyruvate route which is preferentially utilized for the
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Nat. Prod. Rep., 1999, 16, 565–574
formation of the prenyl chain of tetrahydromenaquinone, an
essential metabolite synthesized during the exponential growth
phase, as well as the MVA pathway, which is utilized for the
biosynthesis of the monoterpenic moiety of naphterpin produced during the stationary phase.72 A similar dichotomy was
found in an Actinoplanes sp. in which the prenyl chain of
tetrahydromenaquinone-9 resulted from the non-mevalonate
pathway and the monoterpenic moiety linked to a benzoquinone
antibiotic BE-40644 was formed via MVA.73 Both pathways
were present in these actinomycetes, but they were apparently
not simultaneously utilized.
Unambiguous identifications of the MVA route are very few
for prokaryotes.25 The enzymic activities of the MVA pathway
were clearly characterized in only two eubacteria: Staphylococcus carnosus and Myxococcus fulvus.27 For the latter microorganism, the presence of the MVA route was confirmed by the
labeling pattern found after incorporation of [1-13C]acetate.63
Incorporation of 13C labeled mevalonate into the carotenoid
zeaxanthin of a bacterium provisionally classified as a Flavobacterium sp.74 or of 13C labeled acetate into verrucosan-2bol, a diterpene from Chloroflexus aurantiacus, gave direct
evidence for the presence of the MVA route in these
organisms.75 In the few investigated Archaea, labeling experiments performed with 13C labeled acetate or deuterium labeled
glycerol or MVA showed that the biphytanyl chain of the lipids
of the thermoacidophilic Caldariella (Sulfolobus) acidophila 76
and the phytanyl chains of the phospholipids of the halophilic
Halobacterium cutirubrum,77 Halobacterium halobium78 and
Haloarcula japonica79 were all derived from MVA. Information on the two metabolic routes for isoprenoid biosynthesis is
still too scarce to draw any definitive conclusions on their
distribution amongst prokaryotes.
The GAP/pyruvate route is however widely distributed
amongst eubacteria. It was found in many opportunistic
pathogens as well as in innocuous species closely related to well
known pathogenic bacteria for man.80 This implies that the
enzymes involved in this metabolic route represent targets for
the design of new types of antibacterial drugs that are highly
required in order to overcome antibiotic resistance which is
becoming a rather serious problem.
5 The distribution of the GAP/pyruvate pathway
amongst phototrophic eukaryotes
5.1 Essential chloroplast isoprenoids and sterols from
green algae
The failure to efficiently incorporate 14C labeled acetate or
MVA into plant carotenoids and to block their biosynthesis with
the HMG-CoA reductase inhibitor mevinolin suggested the
possible presence of an alternative route for the formation of
these isoprenoids.6,7 In contrast, CO2 or pyruvate efficiently
labeled carotenoids in maize or spinach chloroplasts.81–84
Furthermore, mono- and diterpenes were mostly only weakly
labeled from acetate or from MVA.25 Such data, as well as
related contributory information suggested the possible presence of the bacterial GAP/pyruvate route in the chloroplasts.
Indeed, these first two C3 precursors are both derived from
glucose directly resulting from photosynthesis. The same
labeling methods as those which proved successful for bacteria
were utilized with phototrophic organisms. 13C Labeled acetate
however had to be replaced by glucose in most experiments. All
algae and plant systems were heterotrophically grown with a
single 13C labeled carbon source under low light intensities that
did not allow photosynthesis. Such conditions were however
still compatible with chloroplast differentiation and consequently with the biosynthesis of their pigments containing
prenyl moieties (carotenoids, phytyl chain of chlorophylls,
plastoquinone). They also avoided possible scrambling of the
label from 13CO2, resulting from recycling after catabolism of
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the 13C labeled carbon source, as a consequence of photosynthesis.
The first successful experiments were performed with the
green alga Scenedesmus obliquus.85 The labeling patterns
observed in all isoprenoids after incorporation of 13C labeled
acetate or glucose isotopomers were identical with those
previously observed in similar experiments performed with
bacteria. They corresponded to the processing of pyruvate and
GAP via the glyoxylate and tricarboxylic acid cycles when
acetate was the carbon source, or via glycolysis when glucose
was utilized (Scheme 3). Surprisingly, all investigated isoprenoids (sterols 18, ubiquinone 12, phytol 14, carotenoids 15
and plastoquinone 16, Fig. 1) were synthesized via the MVA
independent route. No evidence for the MVA route was found.
Identical results were obtained with two other green algae
(Chlamydomonas reinhardtii and Chlorella fusca).64
The same labeling experiments were performed using 13C
labeled glucose with higher plant systems: barley seedlings
(Hordeum vulgare), an axenic culture of a duckweed (Lemna
gibba) and a green tissue culture of carrot (Daucus carotta).86 A
clear dichotomy was observed. Sterols 18 presented, as
expected, the labeling pattern corresponding to the MVA route
with acetyl-CoA formed from pyruvate resulting from glucose
catabolism via glycolysis (Scheme 3). In contrast, the investigated chloroplast isoprenoids (phytol 14, carotenoids 15 and
plastoquinone 16), all derived from GGPP 9 (Scheme 2), were
labeled according to the bacterial MVA-independent route
(Scheme 3). Under the labeling conditions we utilized, neither
scrambling nor superposition of the labeling patterns from the
two routes was observed. The investigated plant systems (two
from monocots, one from a dicot) are probably representative of
all plants, and the clear compartmentation observed in the
biosynthesis of plant isoprenoids emerges as a general feature:
the well known and well investigated MVA route operates in the
cytoplasm, yielding the IPP for the formation of the sterols and
even ubiquinone,87 whereas the recently discovered bacterial
GAP/pyruvate route operates in the chloroplasts giving the IPP
required for the biosynthesis of carotenoids, phytol and
plastoquinone prenyl chain. The same compartmentation was
found in the Rhodophyte Cyanidium caldarium and in the
Chrysophyte Ochromonas danica.64 In the heterotrophically
grown Euglenophyte Euglena gracilis, however, only the MVA
route was found for the formation of ergosterol in the
cytoplasm, as well as for phytol in the chloroplasts.64 The
probable ubiquity of the GAP/pyruvate route is well illustrated
by the biosynthesis of many other isoprenoids of more restricted
distribution and usually of less obvious physiological significance (see section 5.2). We will also see (section 5.3) that
this compartmentation between cytoplasm and chloroplasts is
not always as clear cut as observed in the above described plant
systems: some intermediates (e.g. IPP, GPP, FPP) can be
exchanged.
5.2 Isoprenoids from secondary metabolism
The first proof for the presence of the MVA independent route
in phototrophic eukaryotes was obtained by performing incorporation experiments in Ginkgo biloba embryos with mainly
13C labeled glucose isotopomers in order to elucidate the
biosynthesis of diterpenoids of the ginkgolide 17 (Fig. 1) and
bilobalide series.29,88 These experiments were run independently from those performed on bacterial hopanoids. The same
labeling patterns as those found for bacterial isoprenoids were
observed in the isoprenic units from these ginkgo diterpenoids
and had to be interpreted in the same way. Moreover, as in the
case of the sesquiterpenic pentalenic acid derivatives from
Streptomyces,66 incubation of [U-13C6]glucose also allowed
light to be shed on the formation of the branched C5 skeleton of
isoprenic units in the MVA independent route revealing a
rearrangement step. Indeed, in one of the isoprenic units of the
ginkgolides 17 , an unexpected 1J 13C–13C coupling constant
was observed for the signals of two carbon atoms derived from
C-2 and C-4 of IPP. In the MVA pathway, these carbon atoms
would be derived from two different acetyl-CoA units and no
coupling should be found. In the MVA independent route
however, they both arise from the GAP molecule, and their
signal should appear as previously mentioned as two doublets
with a small 2J coupling because of the transposition. In one of
the ginkgolide isoprenic units, an additional rearrangement
involved in the formation of this diterpenic carbon skeleton
restores the original non-interrupted sequence of the carbon
atoms and is at the origin of the observed 1J 13C–13C
coupling.
Incorporation of 13C labeled glucose into isoprenoids and
analysis of the resulting isoprenoid 13C NMR spectra allowed
the unambiguous characterization of the MVA independent
route in bacteria and in ginkgo embryos. This experimental
protocol opened the way to a whole array of new investigations.
The recognition of DX as an isoprenoid precursor30 has allowed
the completion of such investigations by feeding experiments
using the deuterium or 13C labeled pentulose. Such methods
successfully addressed a variety of unsolved problems related to
isoprenoid biosynthesis in plants and reinforced the operation of
the non-mevalonate pathway in providing numerous plant
isoprenoids. Incubation of leaves or shoots from Salix cinerea,
Populus nigra or Chelidonium majus with deuterium labeled
methyl deoxyxyluloside resulted in deuterium labeling of the
emitted isoprene.89 Feeding of deuterium labeled free DX
showed that volatiles (mono- and sesquiterpenes) emitted by the
leaves of the Lima bean Phaseolus lunatus after jasmonic acid
treatment or spider mite infestation or constitutive volatiles
emitted by leaves of flowers of Eucalyptus globulus, Clematis
vitalba, Hedera helix, Passiflora coerulea and Callicarpa
japonica were essentially derived from the MVA independent
route.90 Incorporation of [1-13C]- and in some cases of [U13C ]glucose showed the non-MVA origin of other terpenoid
6
series: monoterpenes from higher plants91 (Pelargonium graveolens, Mentha 3 piperita, Mentha pulegium and Thymus
vulgaris) and from the liverworts92 (Conocephalum conicum
and Ricciocarpos natans), monoterpenic moiety of the iridoid
glucoside secologanin of Catharanthus roseus,93 sesquiterpenoids in mycorrhized barley roots94 or in the chamomile
Matricaria recutita,95 diterpenoids in cell cultures of the
liverwort Heteroscyphus planus96 and higher plant tissue
cultures from Salvia miltiorrhiza,28 Taxus sinensis,97 and
Marrubium vulgare.98
Whereas isoprene biosynthesis is directly related to chloroplasts, the formation of mono- and diterpenoids is not well
documented. These isoprenoids are believed to be formed in
plastids or plastid related organelles. Their formation via the
GAP/pyruvate route was therefore not completely unexpected.
Finally, the presence of the bacterial MVA independent route in
plant chloroplasts is in full accord with the prokaryotic origin of
these organelles.
5.3 Intermediate exchanges between the mevalonate and
the GAP/pyruvate pathways in plants
Two biosynthetic pathway are operating in plant cells for the
formation of isoprenoids. On the one hand, the MVA route is
located in the cytoplasm and is responsible for the formation of
sterols, triterpenes, many sesquiterpenes and the prenyl chain of
ubiquinone. On the other hand, the alternative MVA independent route is present in the chloroplasts (or in chloroplast related
organelles) and is responsible for the formation of the
terpenoids required for the photosynthetic machinery (carotenoids, phytol, prenyl chain of plastoquinone) and most probably
mono- and diterpenoids. The compartmentation is however not
as clear cut as described above. IPP 5 and the acyclic polyprenyl
diphosphates GPP 7 and FPP 8 (Scheme 2) are common to both
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biosynthetic sites, and evidence is available showing that
exchanges occur at least at the level of IPP 5, GPP 7 and FPP
8.
Feeding experiments with ginkgo embryos using 13C labeled
mevalonate or glucose shed light on a mixed contribution of
both pathways to the formation of ginkgolides. GGPP, the
acyclic C20 precursor of diterpenoids, was essentially (98–99%)
derived from plastidic IPP synthesized via the GAP/pyruvate
pathway. The remaining GGPP (1–2%) showed mixed origin:
four fifth being derived from cytoplasmic FPP and one fifth
from cytoplasmic IPP, both imported from cytoplasm and
consequently synthesized from mevalonate.29 Similar observations are available for the formation of the diterpenoids phytol
and heteroscyphic acid A in cell cultures of the liverwort
Heteroscyphus planus.99–101
Such a mixed origin of isoprenic units was also found for the
formation of the sesquiterpenes bisaboloxide A and chamazulene from Matricaria recutita after injection of [1-13C]glucose
into the anthodia of the plants.96 Their C15 skeleton of FPP was
made from a C10 GPP subunit predominantly formed via the
GAP/pyruvate pathway and completed by a third isoprenic
derived either from the MVA pathway or from the GAP/
pyruvate pathway.
Labeling experiments were performed with lima bean leaves
using either deuterium labeled MVA or DX, and the degree of
labeling of induced volatile terpenoids by jasmonic acid
treatment of spider mite infection was measured by mass
spectrometry.90 Again, a mixed origin was detected. The
monoterpenes ocimene and linalool were mainly synthesized
via the GAP/pyruvate route with however some significant
contribution from the MVA pathway. The sesquiterpenic
derivative, 4,8-dimethylnona-1,3,7-triene, was labeled from
both precursors with intensities of similar orders of magnitude,
with however a preference for MVA, whereas the diterpenoid
4,8,13-trimethyltrideca-1,3,7,11-tetraene was essentially derived from DX, especially in the case of spider mite infection.
A minor contribution from the GAP/pyruvate route was also
found for sterol biosynthesis (normally occurring from MVA)
in the plant cell cultures of Catharanthus roseus.38 Label from
[1-13C]- and [2,3,4,5-13C4]DX was mainly incorporated into
phytol and carotenoids, but was diverted to a minor extent
(about 6%) into sitosterol.
Such a minor contribution of the MVA pathway to the
formation of isoprenoids which are mainly biosynthesised via
the GAP/pyruvate route is probably the most rational explanation for the low, but unambiguous, incorporation of 14C labeled
MVA observed in former studies on the biosynthesis of
carotenoids, mono- and diterpenes.
Overfeeding of metabolites such as MVA or DX, which are
not accumulated by cells, does not represent normal physiological growth conditions. High concentrations of a precursor
might activate the corresponding metabolic pathway and
consequently lead to an overestimation of its contribution to the
production of an isoprenoid. Such experiments however shed
light on the main route utilized for the biosynthesis of an
isoprenoid and on the possible exchanges between the two
routes. Feeding of a more ‘neutral’ precursor such as glucose is
probably preferable, although normal growth conditions for
phototrophic organisms would require carbon dioxide as sole
carbon source.
These labeling experiments clearly showed that exchanges of
common metabolites (IPP, GPP and FPP) are possible between
the two pathways utilized by plants for isoprenoid biosynthesis.
Even if no clear data are available on the site of biosynthesis of
most secondary metabolites of terpenic origin, the superposition
of two different labeling patterns indicates a probable active
transport process for prenyl diphosphate between cytoplasm
and chloroplasts. The sites for isoprenoid biosynthesis should
therefore be clearly identified in the future and this represents a
major target for the next few years.
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Nat. Prod. Rep., 1999, 16, 565–574
Finally, the presence of two different biosynthetic routes
leading to the same metabolite, IPP, offers a mechanism for
plants to regulate isoprenoid biosynthesis. The two starter
metabolites of the non-MVA route, GAP and pyruvate, are
directly derived from photosynthesis. Accordingly, this metabolic route is probably more rapidly implemented than the
MVA pathway. The exploration of the respective contributions
of both pathways to isoprenoid formation will open up a new
research theme in plant physiology.
6
Conclusion
Further development of the elucidation of the MVA independent route (Scheme 4) will focus on the identification of the
remaining intermediates and enzymes involved in the conversion of MEP into IPP. Formally, the conversion of DXP into IPP
requires at least one phosphorylation, three reductions and the
elimination of two water molecules. Incubation of deuterium
labeled precursors should allow the determination of the origin
of the IPP protons, yielding possible clues for the identification
of such intermediates. Incubation of [6,6-2H2]glucose into
ubiquinone from E. coli30 or into the hopanoids of Zymomonas
mobilis102 has shown that the deuteriums on carbon atoms
derived from C-1 of IPP and accordingly from C-3 of GAP and
C-6 of glucose are retained, whatever the catabolic pathway
utilized for the catabolism of the hexose. This was confirmed by
the incubation of [1,1,4,4-4H2]ME with E. coli.55 Only two
isotopomers of ubiquinone with respectively four and eight
deuteriums were detected by mass spectrometry next to
unlabeled ubiquinone. This was in accord with the former
labeling experiments with [6,6-2H2]glucose and suggested in
addition that no changes occurred in the oxidation state of
carbon atoms corresponding to C-4 of IPP when ME was
incorporated into isoprenoids. Incubation of [1,1,1-2H3]DX
with E. coli and NMR analysis of the resulting quinone showed
that the three deuterium atoms of DX were all retained in the
ubiquinone methyl groups.103 Further, when [3-2H]DX was
incubated with the same bacterium, the deuterium was retained,
whereas after feeding of [4-2H]DX, this deuterium was found
only in the isoprenic unit corresponding to DMAPP and lost in
all those derived from IPP.104 Incubations of deuterium labeled
glucose isotopomers into phytol of the cyanobacterium Synechocystis sp. UTEX 2470 partially confirmed the previous
observations.105 A complete picture is however not available at
present providing a clear overview of the biosynthetic pathway.
With the exception of the reduction step catalyzed by the DXP
reducto-isomerase, no information is available on the other
reduction steps.
The first common enzyme of the two pathways is probably
the IPP isomerase which interconverts IPP and DMAPP
(Scheme 1). Incorporation of 13C and 14C labeled pyruvate into
the monoterpenes of secretory peppermint cells (which are
synthesized via the non-MVA route) in the presence of the IPP
isomerase inhibitor 2-(dimethylamino)ethyl diphosphate resulted in the accumulation of IPP and not DMAPP.106 This
suggested that it is IPP (and not DMAPP) which is formed from
DXP and MEP.
More work is therefore required. The methods employing
incorporation of precursors labeled with stable isotopes such as
13C or 2H are much too tedious, time consuming and suffer from
poor sensitivity. More rapid methods based on molecular
biology and gene identification will give much more rapidly a
better picture of the distribution of the metabolic routes
involved in isoprenoid biosynthesis once.
The formation of DXP is certainly not the first committed
step of the mevalonate-independent pathway. Indeed, DXP or
free DX are key intermediates of other metabolic routes: the
former is the precursor of thiamine diphosphate, the latter of
pyridoxol.34–36 MEP is the first intermediate presenting the
isoprenic motif. Its branched skeleton is formed by the same
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reaction sequence as those involved in the formation of IPP in
the alternative biosynthetic route in the bacterium Corynebacterium ammoniagenes54 as well as in the higher plant
Liriodendron tulipifera.56 MEP, like IPP, can be considered as
a true hemiterpene. If MEP or ME are not involved in other
metabolic routes, the rearrangement catalyzed by the DXP
reducto-isomerase might represent the first committed step of
the MVA-independent pathway, much as the HMGCoA
reductase is the committed step of the MVA route.
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7
Acknowledgments
I thank all my partners and students. The names of many of them
are mentioned in the references. Their contribution to the
chemistry and the biochemistry of bacterial hopanoids during
the last fifteen years made possible the discovery of the MVA
independent pathway. Part of this work was the result of
collaborations with the groups of Professor H. Sahm (Jülich,
FRG) and Professor H. K. Lichtenthaler (Karlsruhe, FRG).
Support was provided by the ‘Centre National de la Recherche
Scientifique’, from 1993 to 1996 by the European Union in the
frame of the project ‘Biotechnology of Extremophiles’ and from
1997 by the ‘Institut Universitaire de France’.
8
References
1 J. D. Connolly and R. A. Hill, in Dictionary of Terpenoids, Chapman
and Hall, New York, 1992.
2 S. L. Spurgeon and J. W. Porter, in Biosynthesis of Isoprenoid
Compounds, ed. J. W. Porter and S. L. Spurgeon, John Wiley and Sons,
New York, 1981, vol. 1, p. 1, and references cited therein.
3 N. Qureshi and J. W. Porter, in Biosynthesis of Isoprenoid Compounds,
ed. J. W. Porter and S. L. Spurgeon, John Wiley and Sons, New York,
1981, vol. 1, p. 47, and references cited therein.
4 H. Kleinig, Annu. Rev. Plant Physiol., 1989, 40, 39.
5 J. Chappel, Annu. Rev. Plant Physiol., 1995, 46, 521.
6 T. J. Bach, Lipids, 1995, 30, 191.
7 H. K. Lichtenthaler, M. Rohmer and J. Schwender, Plant Physiol.,
1998, 101, 643.
8 K. J. Treharne, E. I. Mercer and T. W. Goodwin, Biochem. J,, 1966, 99,
239.
9 D. V. Banthorpe, B. V. Charlwood and M. J. O. Francis, Chem. Rev.,
1972, 72, 115.
10 T. J. Bach and H. K. Lichtenthaler, in Ecology and Metabolism of Plant
Lipids, ed. G. Fuller and W. D. Nes, Am. Chem. Soc. Symp. Series,
1987, 325, American Chemical Society, Washington DC, p. 109.
11 T. J. Bach, A. Motel and T. Weber, Rec. Adv. Phytochem., 1990, 24,
1.
12 K. Kreuz and H. Kleinig, Planta, 1981, 153, 578.
13 K. Kreuz and H. Kleinig, Eur. J. Biochem., 1984, 141, 531.
14 F. Lütke-Brinkhaus and H. Kleinig, Planta, 1987, 171, 406.
15 M. Rohmer, P. Bouvier-Navé and G. Ourisson, J. Gen. Microbiol.,
1984, 130, 1137.
16 M. Rohmer, Pure Appl. Chem., 1993, 65, 1293.
17 G. Ourisson and M. Rohmer, Acc. Chem. Res., 1992, 25, 403, and
references cited therein.
18 K. Poralla, T. Härtner and E. Kannenberg, FEMS Microbiol. Lett.,
1984, 23, 253.
19 M. A. F. Hermans, B. Neuss and H. Sahm, J. Bacteriol., 1991, 173,
5592.
20 G. Kleemann, G. Alskog, A. M. Berry and K. Huss-Danell,
Protoplasma, 1994, 183, 107.
21 E. L. Kannenberg, M. Perzl and T. Härtner, FEMS Microbiol. Lett.,
1995, 127, 255.
22 G. Flesch and M. Rohmer, Eur. J. Biochem., 1988, 175, 405.
23 M. Rohmer, B. Sutter and H. Sahm, J. Chem. Soc., Chem. Commun.,
1989, 1471.
24 M. Rohmer, M. Knani, P. Simonin, B. Sutter and H. Sahm, Biochem.
J., 1993, 295, 517.
25 M. Rohmer, in Comprehensive Natural Product Chemistry, ed. D. E.
Cane, Pergamon, Oxford, UK, 1999, vol. 2, ch. 3, p. 45, and references
cited therein.
26 G. A. Sprenger, FEMS Microbiol. Lett., 1996, 145, 301.
27 S. Horbach, H. Sahm and R. Welle, FEMS Microbiol. Lett., 1993,
111,135.
28 M. Rohmer, M. Seemann, S. Horbach, S. Bringer-Meyer and H. Sahm,
J. Am. Chem. Soc., 1996, 118, 2564.
29 M. K. Schwarz, Ph D Thesis, Nb ETH 10951, Eidgenössische
Technische Hochschule, Zürich, Switzerland, 1994, and references
cited therein.
30 S. T. J. Broers, Ph D Thesis Nb 10978, Eidgenössische Technische
Hochschule, Zürich, Switzerland, 1994.
31 D. Zhou and R. H. White, Biochem. J., 1991, 273, 627.
32 A. Yokota and K. I. Sasajima, Agric. Biol. Chem., 1984, 48, 149.
33 A. Yokota and K. I. Sasajima, Agric. Biol. Chem., 1986, 50, 2517.
34 S. David, B. Estramareix, J.-C. Fischer and M. C. Thérisod, J. Am.
Chem. Soc., 1981, 103, 7351.
35 K. Himmeldirk, B. G. Sayer and I. D. Spenser, J. Am. Chem. Soc.,
1998, 120, 3581, and references cited therein.
36 K. Himmeldirk, I. A. Kennedy, R. E. Hill, B. G. Sayer and I. A.
Spenser, Chem. Commun., 1996, 1187.
37 S. Rosa Putra, L.-M. Lois, N. Campos, A. Boronat and M. Rohmer,
Tetrahedron Lett., 1998, 39, 23.
38 D. Arigoni, S. Sagner, C. Latzel, W. Eisenreich, A. Bacher and M. H.
Zenk, Proc. Natl. Acad. Sci. USA, 1997, 94, 10600.
39 G. A. Sprenger, U. Schörken, T. Wiegert, S. Grolle, A. A. de Graaf,
S. V. Taylor, T. P. Begley, S. Bringer-Meyer and H. Sahm, Proc. Natl.
Acad. Sci. USA, 1997, 94, 12857.
40 L.-M. Lois, N. Campos, S. Rosa Putra, K. Danielsen, M. Rohmer and
A. Boronat, Proc. Natl. Acad. Sci. USA, 1998, 95, 2105.
41 B. M. Lange, M. R. Wildung, D. McCaskill and R. Croteau, Proc. Natl.
Acad. Sci. USA, 1998, 95, 2100.
42 D. L. Turner, H. Santos, P. Fareleira, I. Pacheco, J. Le Gall and A. V.
Xavier, Biochem. J., 1992, 285, 387.
43 D. Ostrovsky, A. Shashkov and A. Sviridov, Biochem. J., 1993, 295,
901.
44 D. Ostrovsky, E. Kharatian, T. Dubrovsky, O. Ogrel, I. Shipanova and
L. Sibeldina, BioFactors, 1992, 4, 63.
45 D. Ostrovsky, E. Kharatian, I. Malarova, I. Shipanova, L. Sibeldina, A.
Sashkov and G. Tantsirev, BioFactors, 1992, 3, 261.
46 S. W. Shah, S. Brandänge, D. Behr, J. Damén, S. Hagen and T.
Anthonsen, Acta Chem. Scand., 1976, B30, 903.
47 R. W. Schramm, B. Tomaszewska and G. Peterson, Phytochemistry,
1979, 18, 1393.
48 R. Kringstad, A. Olsvik Singsaas, G. Rusten, G. Baekkemoen, B.
Smestad Paulsen and A. Nordal, Phytochemistry, 1980, 19, 543.
49 J. de Pascual Terresa, J. C. Hernandez Aubanell, A. San Feliciano and
J. M. Miguel del Corral, Tetrahedron Lett., 1980, 21, 1359.
50 C. W. Ford, Phytochemistry, 1981, 20, 2019.
51 P. Dittrich and S. J. Angyal, Phytochemistry, 1988, 27, 935.
52 J. Kitajima and Y. Tanaka, Chem. Pharm. Bull., 1993, 41, 1667.
53 A. A. Ahmed, M. H. Abd El-Razek, E. Abu Mostafa, H. J. Williams,
A. I. Scott, J. H. Reibenspies and T. J. Mabry, J. Nat. Prod., 1996, 59,
1171.
54 T. Duvold, J.-M. Bravo, C. Pale-Grosdemange and M. Rohmer,
Tetrahedron Lett., 1997, 38, 4769.
55 T. Duvold, P. Calì, J.-M. Bravo and M. Rohmer, Tetrahedron Lett.,
1997, 38, 6181.
56 S. Sagner, W. Eisenreich, M. Fellermeier, C. Latzel, A. Bacher and
M. H. Zenk, Tetrahedron Lett., 1998, 39, 2091.
57 T. Kuzuyama, S. Takahashi, W. Watanabe and H. Seto, Tetrahedron
Lett., 1998, 39, 4509.
58 S. Takahashi, T. Kuzuyama, H. Watanabe and H. Seto, Proc. Natl.
Acad. Sci. USA, 1998, 95, 9879.
59 Y. Shigi, J. Antimicrob. Chemother., 1989, 24, 131, and references
cited therein.
60 T. Kuzuyama, T. Shimizu, S. Takahashi and H. Seto, 1998,
Tetrahedron Lett., 1998, 39, 7913.
61 J. Zeidler, J. Schwender, C. Müller, J. Wiesner, C. Weidemeyer, E.
Beck, H. Jomaa and H. K. Lichtenthaler, Z. Naturforsch., 1998, 53c,
980.
62 S. Pandian, S. Saengchan and T. S. Raman, Biochem. J., 1981, 196,
675.
63 S. Rosa Putra, A. Disch, J.-M. Bravo and M. Rohmer, FEMS
Microbiol. Lett., 1998, 164, 169.
64 A. Disch, J. Schwender, C. Müller, H. K. Lichtenthaler and M.
Rohmer, Biochem. J., 1998, 333, 381.
65 P. J. Proteau, J. Nat. Prod., 1998, 61, 841.
66 D. E. Cane, T. Rossi, A. M. Tillman and J. P. Pachtlako, J. Am. Chem.
Soc., 1981, 103, 1838.
67 N. Orihara, K. Furihata and H. Seto, J. Antibiot., 1997, 50, 979.
68 N. Orihara, T. Kuzuyama, S. Takahashi, K. Furihata and H. Seto,
J. Antibiot., 1998, 51, 676.
69 S. M. Li, S. Hennig and L. Heide, Tetrahedron Lett., 1998, 39,
2717.
70 K. Endler, U. Schuricht, L. Hennig, P. Wetzel, U. Holst, W. Aretz, D.
Böttger and G. Huber, Tetrahedron Lett., 1998, 39, 13.
Nat. Prod. Rep., 1999, 16, 565–574
573
Downloaded on 23 September 2012
Published on 01 January 1999 on http://pubs.rsc.org | doi:10.1039/A709175C
View Online
71 K. Irie, Y. Nakagawa, S. Tomimatsu and H. Ohigashi, Tetrahedron
Lett., 1998, 39, 7929.
72 H. Seto, H. Watanabe and K. Furihata, Tetrahedron Lett., 1996, 37,
7979.
73 H. Seto, N. Orihara and K. Furihata, Tetrahedron Lett., 1998, 39,
9497.
74 G. Britton, T. W. Goodwin, W. J. S. Lockley, A. P. Mundy and N. J.
Patel, J. Chem. Soc., Chem. Commun., 1979, 27.
75 C. Rieder, G. Strauß, G. Fuchs, D. Arigoni, A. Bacher and W.
Eisenreich, J. Biol. Chem., 1998, 273, 18099.
76 M. de Rosa, A. Gambacorta and B. Nicolaus, Phytochemistry, 1980,
19, 791.
77 N. Moldoveanu and M. Kates, Biochim. Biophys. Acta, 1988, 960,
164.
78 K. Kakinuma, M. Yamagishi, H. Fijimoto, N. Ikekawa and T. Oshima,
J. Am. Chem. Soc., 1988, 110, 4861.
79 T. Eguchi, M. Morita and K. Kakinuma, J. Am. Chem. Soc., 1998, 120,
5247.
80 M. Rohmer, Progr. Drug Res., 1998, 50, 136.
81 T. W. Goodwin, Biochem. J., 1958, 68, 26.
82 T. W. Goodwin, Biochem. J., 1958, 70, 612.
83 D. Schulze-Siebert and G. Schultz, Plant Physiol., 1987, 84, 1233.
84 A. Heintze, J. Görlach, C. Leuschner, P. Hoppe, P. Hagestein, D.
Schulze-Siebert and G. Schultz, Plant Physiol., 1990, 93, 1121.
85 J. Schwender, M. Seemann, H. K. Lichtenthaler and M. Rohmer,
Biochem. J., 1996, 316, 73.
86 H. K. Lichtenthaler, J. Schwender, A. Disch and M. Rohmer, FEBS
Lett., 1997, 400, 271.
87 A. Disch, A. Hemmerlin, T. J. Bach and M. Rohmer, Biochem. J.,
1998, 331, 615.
88 M. Schwarz and D. Arigoni, in Comprehensive Natural Product
Chemistry, ed. D. E. Cane, Pergamon, Oxford, 1999, vol. 2, ch. 14, p.
367.
574
Nat. Prod. Rep., 1999, 16, 565–574
89 J. G. Zeidler, H. K. Lichtenthaler, H. U. May and F. W. Lichtenthaler,
Z. Naturforsch., Teil. G, 1997, 52, 15.
90 J. Piel, J. Donath, K. Bandemer and W. Boland, Angew. Chem., Int.
Ed., 1998, 37, 2478.
91 W. Eisenreich, S. Sagner, M. H. Zenk and A. Bacher, Tetrahedron
Lett., 1997, 38, 3889.
92 A. Contin, R. van der Heijden, A. W. M. Lefeber and R. Verpoorte,
FEBS Lett., 1998, 434, 413.
93 K.-P. Adam, R. Thiel, J. Zapp and H. Becker, Arch. Biochem. Biophys.,
1998, 354, 181.
94 W. Maier, B. Schneider and D. Strack, Tetrahedron Lett., 1998, 39,
521.
95 K.-P. Adam and J. Zapp, Phytochemistry, 1998, 48, 953.
96 K. Nabeta, T. Saitoh, K. Adachi and K. Komura, Chem. Commun.,
1998, 671.
97 W. Eisenreich, B. Menhard, P. J. Hylands, M. H. Zenk and A. Bachert,
Proc. Natl. Acad. Sci. USA, 1996, 93, 6431.
98 W. Knöss, B. Reuter and J. Zapp, Biochem. J., 1997, 326, 449.
99 K. Nabeta, T. Kawae, T. Kikuchi, T. Saitoh and H. Okuyama, J. Chem
Soc., Chem. Commun, 1995, 2529.
100 K. Nabeta, T. Ishikawa, T. Kawae and H. Okuyama, J. Chem. Soc.,
Chem. Commun., 1995, 681.
101 K. Nabeta, T. Kawae, T. Saitoh and T. Kikuchi, J. Chem. Soc., Perkin
Trans. 1, 1997 261.
102 T. Duvold, Ph D Thesis, Université Louis Pasteur, Strasbourg, France,
1997.
103 J.-L. Giner and B. Jaun, Tetrahedron Lett., 1998, 39, 8021.
104 J.-L. Giner, B. Jaun and D. Arigoni, Chem. Commun., 1998, 1857.
105 P. J. Proteau, Tetrahedron Lett., 1998, 39, 9373.
106 M. McCaskill and R. Croteau, Tetrahedron Lett., 1999, 40, 653.
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