04 Terpenoides Compilation
04 Terpenoides Compilation
04 Terpenoides Compilation
Plants contain an enormous range of isoprenoid compounds with a wide variety of structures and functions. Some are
primary metabolites, e.g., the steroids, but the majority are synthesized as secondary metabolites that are uniquely plant
products.
Isoprenoids have been known since antiquity as ingredients of perfumes, soaps, flavoring and as food colorants.
The isoprenoids are built up of C5 isoprene units and the nomenclature of the main classes reflects the number of isoprene
units present (Table 1).
Table 1. Main Classes of Isoprenoids found in Plants
IPP DMAPP
Monoterpenoids GPP
HMG-CoA O HO O
O HO reductase
HO H
HO OH
NADP NADPH mevaldic acid
(3R)-mevalonic acid
ATP
mevalonate-5-diphosphate
decarboxylase
ADP
2
O3PO
O O O
O O
O P O P O
3 O O P O P O
O O CO2, PO4
isopentenyl pyrophosphate (IPP) O O
DXP PATHWAY
Recently a second pathway to isopentenyl pyrophosphate was discovered and in 2002 this pathway was shown to occur in
grapes.
Pyruvate reacts with thiamine diphosphate (TPP) to form active acetaldehyde (TPP-C2). See Biochemistry of Yeast
Fermentation; Sugars; Alcoholic Fermentation. Active acetaldehyde then reacts with glyceraldehyde-3-phosphate to form
DXP. See Figure 2.
COOH HO H
O H OP
OH
OH
R N S
R glyceraldehyde 3-phosphate
R S N S
N from glycolysis
OPP
OPP OPP
H OH
O
OH
OP
OP OH
R N S
O OH
1-deoxy-D-xylulose 5-phosphate
DXP
OPP
FIGURE 2
OH OH
OP OP OP OP
OP
O OH OH OH OH O OH
1-deoxyxylulose 2-C-methyl-D-erythritol 1,4-dihydroxy-3-methylbutan-2-one
isopentenyl diphosphate
5-phosphate 4-phosphate 1-phosphate
IPP
DXP MEP
Conversion of DXP to Isopentenyl pyrophosphate
H
O O
pinacol-like OH OH
rearangement
OP OP OP
DXP OP
O OH reductoisomerase HO O OH OH OH
OH
H 2-C-methyl-D-erythritol
1-deoxy-D-xylulose 5-phosphate 4-phosphate
DXP MEP
CDP-ME
CTP
synthase
O
NH2 NH2
O P OH
OH N N
O O CDP-ME OH O O
kinase
O P O P O N O O P O P O N O
O O
OH OH OH OH OH OH OH OH
HO OH HO OH
4-(CDP)-2-C-methyl-D-erythritol 2-phosphate 4-(CDP)-2-C-methyl-D-erythritol
CDP-ME2P CDP-ME
O OH
O O O
P
HO P
OP OP OP OP
O OH
H OH O OH OH OH OH
OH
2-C-methyl-D-erythritol- steps to be determined
2,4-cyclodiphosphate
OP
OP
dimethylallyl diphosphate isopentenyl diphosphate
DMAPP IPP
FIGURE 3
IPP is isomerized to dimethylallyl pyrophosphate (DMAPP) with IPP isomerase. The isomerization involves addition of a
proton to the sp2 carbon of isopentenyl pyrophosphate and generation of the more stable tertiary carbocation. Elimination of
a proton yields the most stable alkene (trisubstituted, Zaitevs Rule).
B
HS HR H
HS HR PPO
PPO IPP isomerase
PPO
Next IPP reacts with DMAPP to give geranyl pyrophosphate (GPP). In the first step of the reaction, IPP acts as a
nucleophile and displaces a pyrophosphate group from DMAPP. Pyrophosphate is an excellent leaving group. Its four OH
groups have pKa values of 0.9, 2.0, 6.6, and 9.4. Therefore three of the four groups will be primarily in their basic forms at
physiological pH (pH 7.3). A proton is removed in the next step, again yielding the most stable alkene. GPP rearranges to
give the monoterpenes. GPP reacts with a second molecule of IPP to produce farnesyl pyrophosphate (FPP). FPP is the
precursor to the sesquiterpenoids, and the steroids. FPP reacts with a third molecule of IPP to produce geranylgeranyl
pyrophosphate (GGPP). GGPP is the precursor of the carotenoids.
prenyl transferase
OPP OPP OPP
OPP
DMAPP HS
HS geranyl pyrophosphate (GPP)
IPP B
IPP
prenyl transferase
OPP
IPP
prenyl transferase
OPP
Interest in the monoterpenes originated due to their use in perfumes and as food flavors. About forty monoterpene
compounds have been identified in grapes. Some of the monoterpene alcohols have a strong odor, especially geraniol, nerol,
citronellol, linalol, -terpineol, wine lactone, rose oxide and hotrienol, which has a floral aroma reminiscent of rose essence.
The olfactory perception thresholds of these compounds are rather low, as little as a few hundred micrograms per liter. The
most odoriferous are rose oxide and wine lactone. Furthermore, the olfactory impact of monoterpene compounds is
synergistic. Geraniol and linalol play an important role in the aromas of grapes and wines from the Muscat family (Muscat
Petits Grains, Muscat of Alexandria, Muscat of Ottonel and Muscat dAlsace), as concentrations are often well above the
olfactory perception thresholds.
These compounds also play a role in the Muscat aroma of some Alsatian and German grape varieties: Gewrztraminer (rose
oxide and wine lactone), Pinot Gris, Riesling, Auxerrois, Scheurebe, MuIler-Thurgau, etc. However, monoterpenes are only
partially responsible for the varietal aromas of these wines and do not explain all of the nuances.
Figure 4 shows the biosynthesis of the important monoterpenes skeletons found in grapes.
OH
OH OH
OH
OH
O
O
OH O rose oxide
hotrienol -terpineol wine lactone
As with the anthocyanidins, most of the monoterpenes found in grape juice are present as glycosides. The main glycoside is
the glucoside (i.e. -D-glucopyranose attached to the monoterpene) but three disaccharides are also present: 6-O--L-
arabinofuranosyl--D-glucopyranose, 6-O--rhamnosyl--D-glucopyranoside and 6-O--D-glucopyranoside.
OH O HO O
O O
HO O HO O
OH HO monoterpene OH OH HO monoterpene
HO OH OH
6-O--L-Arabinofuranosyl--D-glucopyranoside 6-O--Apiofuranosyl--D-glucopyranoside
O OH
HO O
O O
HO monoterpene HO
HO
HO OH HO monoterpene
OH OH
6-O--L-Rhamnopyranosyl--D-glucopyranoside -D-glucopyranoside
Formation of the Major Monoterpenes in Wine from Geranyl pyrophosphate
OH OH
geraniol citronellol
OPP
OPP
HS
OPP OH
PPO IPP
OPP
OH
linalool
linalyl cation
OH
HO O
FIGURE 4.
CAROTENOIDS
The carotenoids are an abundant group of naturally occurring pigments, present in all green tissues, where they are
constituents of the chloroplast, as well as being responsible for most of the yellow to red colors of flowers and fruits.
Carotenoid hydrocarbons are called carotenes, whereas derivatives containing oxygen functions are the xanthophylls.
Unripe green fruit contains the same pigments as other photosynthetic tissues but upon ripening the chloroplasts
differentiate into chromoplasts (storage organelles for pigments) and there is often, but not always, de novo synthesis of
carotenoids.
In the grape, the concentrations of the carotenoids decrease steadily from bloom on, and at the same time the concentrations
of the C13-norisoprenoids steadily increase. Exposure of grapes to sunlight during ripening accelerates carotenoid
breakdown and in accompanied by an increase in the glycosylated C13-norisoprenoid derivative content (glycosides).
The most important carotenoids in grapes, in decreasing order, are lutein, -carotene, neoxanthin and lutein-5,6-epoxide.
OH
lutein
HO
-carotene
C neoxanthin
O
OH OH
HO
OH
lutein-5,6-epoxide
O
HO
The first dedicated step in the formation of carotenoids is the head-to-head condensation of two molecules of all-trans
geranylgeranyl pyrophosphate (GGPP) via the cyclopropyl carbonyl pyrophosphate, prephytoene pyrophosphate (PPPP) to
form phytoene. See Figure 5.
The Formation of 15-cis-phytoene from GGPP
PPO
OPP
GGPP GGPP
OPP
PPPP
OPP
H
H
15-cis-phytoene
FIGURE 5.
The sequence from phytoene to lycopene involves four stepwise dehydrogenations, occurring alternatively to either side of
the chromophore to form phytofluene, -carotene, neurosporene and lycopene. In addition, in higher plants, the 15-15
double bond is isomerized from the cid to the trans configuration. See Figure 6.
15-cis-phytoene
phytoene desaturase
15-cis-phytofluene
all trans-phytofluene
phytoene desaturase
-carotene
-carotene desaturase
neurosporene
-carotene desaturase
lycopene
FIGURE 6.
The cyclohexane-ring end-groups on many of the carotenoids are formed independently.
Lycopene
Nu Enzyme
H Lycopene
Nu Enzyme Nu Enzyme
Nu Enzyme
-carotene
OH
zeaxanthin
HO
OH
violaxanthin O
O
HO
FIGURE 7.
-Carotene can result from the dehydration and enzyme release step taking place at one of the cyclohexane end-groups via
the removal of a proton on the other side of the carbon which has been attacked by the enzymic nucleophile.
Nu Enzyme
H Lycopene
Nu Enzyme
Enzyme Nu H
Nu Enzyme
-carotene
OH
Lutein
HO
FIGURE 8.
C13-NORISOPRENOIDS
A vast number of apparently carotenoid-derived substances have been identified in plant extracts, but knowledge of the
biochemistry of carotenoid catabolism is still extremely limited. The in vivo cleavage of the carotenoid chain is generally
considered to be catalyzed by dioxygenase systems. Although all the in-chain double-bonds seem to be vulnerable to
enzymatic attack, thus resulting in the formation of major fragment classes with 10, 13, 15 or 20 carbon atoms, in fruit
tissues a bio-oxidative cleavage of the 9,10 (9',10') double bond seems to be the most preferred. This last cleavage reaction
creates a large number of aroma products.
' 4'
3'
5
' ' ' '
' 14
' 12 ' 10 ' 8 '
15 13 11 2'
1 7 9 11 13 15 9 7 6'
2 6 8 10 12 14 1'
3 5
4
C20 (Retinoids)
C15 (Plant hormones)
The three essential steps for the formation of C13-norisoprenoid aroma compounds can therefore be considered to consist of
(i) the initial dioxygenase cleavage,
(ii) the subsequent enzymatic transformation(s) of the primary degradation product in natural tissues, and
(iii) formation of the flavorless glycoside.
During winemaking, acid-catalyzed conversion of the glycoside into an aroma compound occurs. Figure 9 shows how the
carotenoid, neoxanthin may yield two C13-norisoprenoid glycosides in this way, which during the winemaking process
produce the two powerful odorants, vitispirane and damascenone.
Glycosylation of any secondary metabolite will significantly increase its water solubility and may occur for a number of
reasons. Some of these may be for
(i) accumulation and storage,
(ii) transport,
(iii) protect the plant from any toxicity of the liphophilic compounds (e.g., the cyanogenic glycosides are hydrolyzed by -
glucosidase releasing toxic cyanide as a defense mechanism),
(iv) stability of compound (the anthocyanidins are more stable as their glycosides, the anthocyanins).
Formation of Two C13-Norisoprenoid Glycosides from the Carotenoid Neoxanthin
C
O
OH OH
HO
neoxanthin carotenoid cleavage enzymes
O
C
O
OH O
HO
OH
grasshopper ketone
epoxide ring opening
enzymatic reduction
O
OH
C OH
HO
OH OH
HO
enzymatic reduction
allenic triol
glucosylation OH
O OH
C OH HO
OH O
HO glucosylation
OH
HO HO
OH OH
HO
OH
O
winemaking OH
HO
process
O
O
winemaking
process
damascenone
vitispirane
FIGURE 9.
Pure Appl. Chem., Vol. 71, No. 12, pp. 22792284, 1999.
Printed in Great Britain.
q 1999 IUPAC
Michel Rohmer
Universite Louis Pasteur/CNRS/Institut Universitaire de France, Institut Le Bel,
4 rue Blaise Pascal, 67070 Strasbourg Cedex, France
INTRODUCTION
Isoprenoids are present in all living organisms. The rst investigations on their biosynthesis were mainly
performed on sterols, with liver tissues, yeast and several plant systems [13]. The C5 isoprenic skeleton
was shown to be derived from the well-known sequence found in all textbooks (Fig. 1, Scheme A),
starting from acetyl-CoA 4 and leading to isopentenyl diphosphate 7 (IPP) via mevalonate 6 (MVA). The
committed step of this biosynthetic route is the reduction of hydroxymethylglutaryl CoA 5 into MVA
catalyzed by the HMGCoA reductase. Mevalonate 6 was unanimously accepted as the universal
isoprenoid precursor in all living organisms, despite some contradictory results mainly obtained when
working on isoprenoid biosynthesis from plant chloroplasts and from bacteria. Feeding experiments using
13
C-labeled carbon sources allowed the detection in bacteria and the partial elucidation of an alternative
route towards IPP (Fig. 1, Scheme B), starting from triose phosphate derivatives, in which 2-C-methyl-D-
erythritol 4-phosphate 10 (MEP) is the rst intermediate presenting the branched isoprenic skeleton. IPP
7 remains, however, a common intermediate to both routes and must therefore be considered as the
universal isoprenoid precursor. This brief contribution is not intended as an extensive survey of the topic.
It summarizes some of the important steps of the discovery and the elucidation of this novel metabolic
route. More details are found in the accompanying references.
*Lecture presented at the 12th International Symposium on Carotenoids, Cairns, Australia, 1823 July 1999, pp.
22052302.
Correspondence: E-mail: [email protected]
2279
2280 M. ROHMER
Fig. 1 Incorporation of [1-13C]-glucose into isoprenoids via the MVA pathway (A) or via the MEP pathway (B).
carbon atoms by a retrobiosynthetic analysis. Such labeling conditions were later extended to most of
the labeling experiments performed, and were quite different from previous labeling studies, which
were made with bacteria grown on complex media. The additional carbon atoms of the
bacteriohopanepolyol side chain were derived from a D-pentose (according to the stereochemistry,
most probably from a D-ribose derivative) linked via its C-5 carbon to the hopane isopropyl group. The
most interesting problem was, however, found in the triterpenic moiety. The observed labeling pattern
found in the hopane isoprene units did not t with that expected from the MVA pathway. The rst
attempts to interpret these results in the frame of the MVA route [5] could not be conrmed by further
incorporations of 13C-labeled glucose isotopomers into the hopanoids 11 from the bacteria Zymomonas
mobilis, Methylobacterium fujisawaense and Alicyclobacillus acidoterrestris and the prenyl side chain
of ubiquinone 12 from Escherichia coli [6].
Isoprenic units were formed in the new metabolic route from two subunits: the rst was derived from
the C-2 and C-3 carbon atoms of pyruvate 3, and the other was D-glyceraldehyde phosphate 2 (GAP)
[6,7]. All labeling patterns resulting from the above-described labeling experiments were consistent with
the formation of pyruvate and GAP from glucose either via the glycolysis or via the EntnerDoudoroff
pathway or from acetate via the tricarboxylic acid and the glyoxylate cycles [5,6]. Incorporation of doubly
Fig. 2 Key isoprenoids for the identication of the MEP pathway: hopanoids (11), ubiquinone (12), ginkgolides
(13), carotenoids (14), phytol (15) and plastoquinone (16).
labeled [4,5-13C2]-glucose into the hopanoids and ubiquinone from Methylobacterium fujisawaense
showed, in addition, from the long-range 13C/13C 2J coupling constants that a rearrangement allowed the
insertion of the C2 subunit derived from pyruvate decarboxylation between the C-2 and C-3 carbon atoms
from GAP [6]. The presence of such a rearrangement was corroborated by the incorporation of [U-13C6]-
glucose into the hopanoids of Zymomonas mobilis [7]. From these data, a hypothetical biogenetic scheme,
which proved later to be veried, was proposed. Similar experiments were performed by Schwarz and
Arigoni on the biosynthesis of diterpenoids of the ginkgolide 13 series in Ginkgo biloba embryos and led
to identical conclusions [8,9].
was applied to labeling experiments performed with phototrophic organisms. A clear dichotomy was
found in higher plants. The MVA route occurred in the cytoplasm and was responsible, as expected, for
the formation of the triterpenoids, including the sterols, whereas the MEP pathway afforded in the
chloroplasts the essential isoprenoids required for the photosynthetic apparatus (carotenoids 14, phytol 15
and plastoquinone 16) [10], as well as the whole series of plastid-related secondary metabolites (isoprene,
mono- and diterpenes) [12,13].
derived from IPP 7 [30]. This observation was corroborated by the incorporation of [3,5,5,5-2H4]-ME
in an E. coli mutant lacking the DXP synthase gene, which solely synthesized its isoprenoid from the
exogenous ME added to the culture medium. Whereas the ubiquinone methyl groups retained their three
deuteriums of ME, the C-3 deuterium was only found in the DMAPP-derived unit and was integrally lost
in those derived from IPP [26].
The conversion of MEP 10 into IPP 7 formally requires the elimination of three molecules of water,
two reduction steps and one phosphorylation. Evidence for a rst reduction was obtained by the discovery
of the DXP isomero-reductase, which introduces a hydride from NADPH on the carbon atom
corresponding to C-4 of IPP. Further evidence for a reduction was obtained by feeding Zymomonas
mobilis with [1-2H]-glucose as the only carbon source [31]. This bacterium utilizes glucose via the
EntnerDoudoroff pathway, is unable to convert pyruvate into GAP and is a strong ethanol producer in
anaerobic growth conditions. C-1 of glucose is completely lost as CO2 by pyruvate decarboxylation and,
accordingly, is not incorporated into the isoprenic units of the hopanoids. This bacterium has no
tricarboxylic cycle. Under such growth conditions, deuterium-labeled NADPH pools are synthesized. In
the triterpenoids of the hopane series, deuterium was found in all carbon atoms derived from C-4 of IPP or
DMAPP, as expected from the DXP isomero-reductase, as well as on all carbon atoms corresponding to
C-2 of DMAPP and IPP. The latter deuterium atom was the signature of an additional reduction step
occurring at an unknown stage of the reaction sequence of the biosynthetic route. The results of this
experiment on the biosynthesis of hopanoids from Zymomonas mobilis shed light on the equivalence of
the isoprenic units, whether they were derived from IPP or from DMAPP, and are consistent with those
obtained with the Catharanthus roseus cell culture [29].
The stereochemistry of the reaction catalyzed by an IPP isomerase [32] and a prenyl transferase [33]
from E. coli has recently been determined. Both enyzmes eliminate the pro R hydrogen from IPP, like all
other known equivalent enzymes. Furthermore, it was shown that the IPP isomerase is not essential in
E. coli: its disruption does not prevent the growth of the bacterium [34]. These data suggest that, if no
other IPP isomerase is present, two distinct routes occur in E. coli, yielding separately IPP 7 and DMAPP
8 from the same intermediate derived from MEP 10.
CONCLUSION
The discovery of a second route for isoprenoid biosynthesis has opened up new elds in isoprenoid
biochemistry. The simultaneous presence of both the MVA and MEP pathways in plant cells and
the exchange of intermediates (IPP, GP, FPP) between the cytoplasm and the plastids indicate the
signicance of this cross-talking [8,15,35,36]. An interesting interpretation is the regulation of the
biosynthesis of volatile isoprenoids released in stress conditions [36].
The presence of the MEP route in bacteria, which are either opportunistic pathogens or closely related
to pathogenic species, opens up a route towards the discovery of enzyme inhibitors, which should
represent novel types of antibacterial agents [37,38]. Finally, the recent nding of the presence of the
two known genes of the MEP pathway in Plasmodium falciparum, the microorganism responsible for
malaria, as well as the growth inhibition of this parasite by fosmidomycin, an inhibitor of the DXP
isomero-reductase, allows new hopes for curing this major disease [39].
REFERENCES
1 S. L. Spurgeon, J. W. Porter. In Biosynthesis of Isoprenoid Compounds (J. W. Porter, S. L. Spurgeon, eds.), vol 1,
pp. 146. John Wiley and Sons, New York (1981).
2 N. Qureshi, J. W. Porter. In Biosynthesis of Isoprenoid Compounds (J. W. Porter, S. L. Spurgeon, eds.), vol. 1,
pp. 4794. John Wiley and Sons, New York (1981).
3 D. A. Bochar, J. A. Friesen, C. V. Stauffacher, V. W. Rodwell. In Comprehensive Natural Products Chemistry.
Isoprenoids Including Carotenoids and Steroids (D. E. Cane, ed.), vol. 2, pp. 1544. Pergamon Press, Oxford,
UK (1999).
4 M. Rohmer. Pure Appl. Chem. 65, 12931298 (1993).
5 G. Flesch, M. Rohmer. Eur. J. Biochem. 175, 405411 (1998).
Mevalonate-independent methylerythritol
phosphate pathway for isoprenoid biosynthesis.
Elucidation and distribution*
Michel Rohmer
*Pure Appl. Chem. 75, 141419 (2003). An issue of reviews and research papers based on lectures presented at the 23rd IUPAC
International Symposium on the Chemistry of Natural Products, Florence, Italy, 28 July2 August 2002 on the theme of natural
products.
E-mail: [email protected]
375
376 M. ROHMER
Fig. 1 Isoprenoids investigated for the elucidation of the MEP pathway: isoprene 1, bacterial hopanoid 2,
ubiquinone 3, menaquinone 4, phytol 5, plastoquinone 6, -carotene 7, ginkgolide 8, sterol 9.
mevalonate pathway, and the identity of the biosynthetic route followed for the biosynthesis of chloro-
plast isoprenoids.
Fig. 2 MVA pathway for isoprenoid biosynthesis with labeling pattern from [1-13C]glucose metabolized via
glycolysis.
14C- and 13C-labeled MVA were respectively incorporated into a sterol of the gliding bacterium
Nannocystis exedens [9] or into the carotenoids of a Flavobacterium sp. [10]. 13C-Labeled acetate
was incorporated into the prenyl moiety of antibiotics of mixed origin from several actinomyces, and
the labeling pattern corresponded to the expected one resulting from the direct incorporation of acetate
into the MVA pathway [11]. Other incorporation attempts led to inconclusive results, which could not
be readily interpreted. Thus, the incorporation of 14C-labeled acetate into the prenyl chain of
ubiquinone from Escherichia coli resulted in a radioactivity distribution, which did not fit with the ex-
pected one from the MVA pathway, and led the authors to propose an alternative variant of this path-
way via acetolactate [12]. 13C-Labeled acetate was not incorporated into this prenyl chain of
ubiquinone, when E. coli was grown on a culture medium containing glucose and amino acid-derived
carbon sources, whereas pyruvate labeled two contiguous carbon atoms derived from C3 and C5 of
DMAPP or IPP [13]. Such results were indeed the premises for the presence of an alternative route for
the formation of isoprene units.
The discovery of a novel pathway for the early steps of isoprenoid biosynthesis was indeed a side-
product of our investigations on the biosynthesis of bacterial triterpenoids of the hopane series (e.g. 2,
Fig. 1) [14]. A C35 skeleton, in which the triterpenic hopane moiety is linked by a carbon/carbon bond
to a polyhydroxylated n-alkyl side chain, characterizes all major bacterial hopanoids [15]. All bacteria
do not produce hopanoids, but in the hopanoid-producing bacteria, such triterpenoids were shown to
play a role of membrane stabilizers, much like sterol do in eukaryotic membranes. Hopanoid concen-
tration is usually of the same order of magnitude as the sterol concentration in eukaryotic cells. This
makes them well suited for investigation by NMR spectroscopy after feeding of 13C-labeled precursors.
Feeding of 13C-labeled acetate to the hopanoid producing bacteria Rhodopseudomonas palustris,
Rhodopseudomonas acidophila, or Methylobacterium organophilum were performed with bacteria
grown on a mineral medium only containing 13C-labeled acetate as the only carbon and energy source
[14]. Such experimental conditions ensured the incorporation of the labeled precursor into the
hopanoids. As most biosynthetic pathways were known, a retrobiosynthetic analysis of the observed la-
beling patterns allowed reconstructing the reaction sequence involved in the formation of the hopanoids.
Indeed, the experiments performed with [1-13C]- and [2-13C]acetate showed that the C5 side chain was
derived from a D-pentose synthesized via the nonoxidative pentose phosphate pathway and linked to the
triterpenic skeleton via its C5 carbon atom. The most interesting problem was, however, found in the
isoprene moiety. The observed labeling pattern completely differed from the expected one from the
MVA pathway. Interpretation was not obvious from the sole experiments performed with labeled ac-
etate. The clues for the elucidation of this metabolic pathway were obtained after incorporation of glu-
cose isotopomers with 13C labeling either at C1, C2, C3, C5, or C6 into the hopanoids of Zymomonas
mobilis [16]. Indeed, Z. mobilis only utilizes glucose as carbon source. This glucose is metabolized via
the EntnerDoudoroff pathway. This bacterium does not possess a complete tricarboxylic acid cycle
and does not convert pyruvate into glyceraldehyde phosphate because of the lack of an active enolase.
From the observed labeling patterns, it could be deduced that isoprene units in the hopanoids were de-
rived from a C2 subunit synthesized via pyruvate decarboxylation and from a C3 subunit corresponding
to a triose phosphate derivative (Fig. 3) [16]. These results were in accordance with the formerly de-
scribed incorporations of 13C-labeled acetate. The observed labeling pattern in the hopanoids from
Fig. 3 MEP pathway for the biosynthesis of isoprenoids with labeling pattern from [1-13C]glucose metabolized via
glycolysis.
Rhodopseudomonas spp. or from M. organophilum simply resulted from the incorporation of acetate
into the glyoxylate cycle, which allows growth on acetate as sole carbon source, and into the tricar-
boxylic acid cycle, yielding finally phosphoenolpyruvate, the precursor of pyruvate and the triose phos-
phate derivatives [16]. They were also confirmed by incorporation of 13C-labeled glucose into iso-
prenoids from bacteria utilizing glucose via the EmbdenMeyerhofParnas pathway and by studies in
other isoprenoid series such as the ubiquitous ubiquinones and menaquinones [16]. Incorporation of
doubly labeled [4,5-13C2]glucose into the hopanoids and ubiquinone of Methylobacterium fujisawaense
and of uniformly labeled [U-13C6]glucose into the hopanoids from Z. mobilis afforded a definitive proof
for the formation of the isoprene units via a C2 and a C3 subunit and shed light on a rearrangement re-
action allowing the insertion of the C2 subunit derived from pyruvate 11 between two carbon atoms
from the triose phosphate-derived C3 subunit [16,17]. This triose phosphate was identified as glycer-
aldehyde phosphate 10 by feeding E. coli mutants lacking each enzymes of the triose phosphate me-
tabolism with either 13C-labeled glycerol in the presence of unlabeled pyruvate, or 13C-labeled pyru-
vate in the presence of unlabeled glycerol [17]. A hypothetical biogenetic pathway was proposed,
including as first step a reaction involving a transketolase-like, thiamin diphosphate-dependent enzyme
catalyzing the condensation of hydroxyethylthiamin derived from pyruvate decarboxylation on the car-
bonyl group of a triose phosphate derivative and a second step with an -ketol rearrangement resem-
bling the reaction catalyzed by the acetolactate isomero-reductase followed by the concomitant reduc-
tion of the resulting aldehyde and yielding methylerythritol 4-phosphate 22 (MEP) [16,17]. MEP
presents the characteristic branched C5 skeleton of all isoprenoids and has to be considered as a
hemiterpene. The pathway was named after this intermediate, which has to date no other known role
than that of an isoprenoid precursor and which most probably is the result of a committed step of the
pathway.
permitting its conversion into IPP (MVA kinase, phosphomevalonate 17 kinase, and diphosphomeval-
onate 18 decarboxylase), random mutations, which were rescued by the addition of MVA to the culture
medium allowed the identification of the ygbP, ychB, and ygbB genes and indicated that they are es-
sential genes of the MEP pathway [4244].
Fig. 4 Independent IPP and DMAPP biosynthesis in the MEP pathway. Incubation of [4-2H]-1-deoxy-D-xylulose
27 or [3-2H]-2-C-methyl-D-erythritol 28.
served, and their signal served as internal reference. The 3-2H was quantitatively retained only in the
DMAPP 20 derived unit, whereas it was completely lost in those derived from IPP 19 (Fig. 4). Such a
different labeling pattern in isoprene units, depending on their origin from IPP or from DMAPP, pointed
to the possible presence of two branches leading separately to the two precursors of isoprene units.
Furthermore, the activity of IPP isomerase is barely detectable in wild-type E. coli, and the deletion of
the idi gene encoding the IPP isomerase does not affect the growth of the bacterium [49]. Definitive proof
of the branching was obtained by a genetic method [50]. The deletion of the dxr gene in E. coli is res-
cued either by the addition of free ME to the culture medium or by the introduction of the genes allow-
ing the utilization of exogenous MVA. When the strain is growing on MVA, it only synthesizes IPP from
MVA and has to rely on a functional protein capable of isomerizing IPP into DMAPP. If in addition the
idi gene is deleted, the novel strain does not grow anymore on MVA, indicating that there is no other way
in E. coli for converting IPP into DMAPP than the reaction catalyzed by the protein encoded by the idi
gene. It grew, however, normally on ME. This shows that IPP and DMAPP can be independently syn-
thesized from MEP derivative by two separate branches: an IPP branch characterized by deuterium loss
from [4-2H]DX or from [3-2H]ME and a DMAPP branch characterized by deuterium retention.
The fate of the 4-H hydrogen of DX can thus be utilized for the detection of a possible branching
of the MEP pathway in plants (Fig. 4). Incubation of a Catharanthus roseus cell culture with
[1-13C, 4-2H]DX resulted in 13C labeling of all isoprene units with no trace of deuterium, suggesting
that no branching occurred, at least in the utilized culture conditions [51]. Feeding of [4-2H]DX to eu-
calyptus twiglets was followed by a slight (0.9 %) but significant deuterium incorporation into the
monoterpene cineol, but only in the DMAPP-derived moiety, with nearly no deuterium found in the iso-
prene unit derived from IPP [52]. Finally, incorporation of [4-2H]DX into the plastid isoprenoids from
a tobacco BY-2 cell culture resulted in the labeling of all isoprene units of phytoene and plastoquinone,
whatever their origin from IPP or from DMAPP was, with the same isotope abundance (ca. 15 %) [53].
These experiments revealed the presence of the branching in plant cells. In C. roseus, only the IPP
branch was detected. In eucalyptus, like in E. coli, both IPP and DMAPP branches were found. In to-
bacco, the DMAPP branch significantly contributed to the formation of all isoprene units. Definitive in-
terpretation of the labeling patterns was only possible once more insight into the reduction reactions in-
volved in the last two steps has been obtained.
tal procedure allowed the detection of intermediates by directly running the 13C NMR spectra on crude
cell-free systems. Additional overexpression of the GcpE gene was followed by the accumulation of
HMBPP 26 [61] and of LytB resulted in the simultaneous formation of IPP 19 and DMAPP 20 [62].
This represented the first indication that the LytB protein was responsible for the conversion of HMBPP
26 into IPP 19 and DMAPP 20 and that the reaction catalyzed by this protein corresponds to the branch-
ing previously described. Finally incubation of 3H-labeled HMBPP with chromoplast from Capsicum
annuum and Narcissus pseudonarcissus led to the formation of radiolabeled carotenoids, confirming the
role of HMBPP 26 as intermediate in the MEP pathway [63].
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Recently, a mevalonate-independent pathway was discovered in bacteria and and C-4 of IPP, and that C-2 and C-3 of pyruvate could
plants that leads to the formation of isopentenyl diphosphate and dimethylallyl supply C-3 and C-5 of IPP in the novel pathway
diphosphate, the two basic precursors of isoprenoids. Although many details (reviewed in Refs 46).
of the widely distributed pathway are unknown, some intermediates, A hypothetical mechanism was suggested with a
mechanisms, enzymes and genes of this novel route have been identified. head-to-head condensation of D-glyceraldehyde 3-
Information on this pathway could provide the basis for the development of phosphate and activated acetaldehyde derived from
new antibiotics, herbicides and antimalarials. pyruvate, resulting in the formation of 1-deoxy-D-
xylulose 5-phosphate as the first precursor of the
Terpenoids and isoprenoids are a structurally diverse novel pathway (S.T.J. Broers, PhD thesis, ETH
group of natural products. More than 25 000 Zrich, 1994; M.K. Schwarz, PhD thesis, ETH Zrich,
representatives with a variety of biological functions 1994) (Fig. 1). This hypothesis was confirmed by
have been reported in the plant kingdom1. To give just efficient incorporation of 2H-labelled 1-deoxy-D-
a few examples, carotenoids serve as light-harvesting xylulose into the isoprenoid side chain of ubiquinone
and light-protecting pigments, sterols play important in E. coli (S.T.J. Broers, PhD thesis, ETH Zrich,
roles as modulators of membrane properties, the 1994). This pentulose had earlier been reported to
phytol side chain of chlorophyll (the most abundant serve as a precursor for the biosynthesis of thiamine7,8
organic pigment) is of terpenoid origin, and a wide and pyridoxal9.
variety of plant terpenoids function as insect
attractants or repellents. Various terpenoids have Deoxyxylulose phosphate pathway is widely
attracted commercial interest as pharmaceuticals or distributed in nature
nutraceuticals. Thus, paclitaxel (Taxol), a diterpene Subsequent isotope incorporation studies using
from yew, has been established as a major cytostatic bacteria, certain algae, plant cell cultures and plant
agent. Lycopene and lutein were recently registered tissues demonstrated the formation of IPP and
as oncopreventive agents. DMAPP from exogenous 1-deoxyxylulose. These
seminal experiments have been reviewed
Pathways of terpenoid biosynthesis recently46,10 and are not covered in detail here. In
All terpenoids are biosynthesized from just two C5 summary (Fig. 2), archaea, certain bacteria, yeasts,
precursors: isopentenyl diphosphate (IPP) and fungi, some protozoa and animals appear to use the
dimethylallyl diphosphate (DMAPP). The mevalonate pathway. However, many bacteria
biosynthesis of these universal terpene precursors via (including many human pathogens), green algae and
mevalonate (Fig. 1) has been elucidated by studies of possibly the malaria parasite Plasmodium
yeast and animal cells (reviewed in Ref. 2). falciparum appear to rely exclusively on the
Radioactivity from labelled mevalonate has been mevalonate-independent pathway. Streptomycetes,
reported to be diverted to a wide variety of plant some algae, mosses and liverworts, marine diatoms,
terpenes, albeit at incorporation rates well below 1%, and higher plants appear to use both pathways.
which were explained by poor uptake of mevalonate Finally, the capacity to biosynthesize isoprenoid
into plant cells or by compartmentalization. precursors appears to be absent in some parasitic
Several results inconsistent with the mevalonate microorganisms that might be able to use terpenoids
pathway led to the discovery of a non-mevalonate from their host.
pathway of terpenoid biosynthesis. For example,
Wolfgang Eisenreich*
Felix Rohdich
unexpected 13C labels were found in hopanoids from Crosstalk between the mevalonate and deoxyxylulose
Adelbert Bacher certain bacteria that had been grown with 13C- phosphate pathways in plants
Lehrstuhl fr Organische labelled acetates3. Furthermore, 13C-labelled glucose In higher plants, the mevalonate route operates in
Chemie und Biochemie, applied to E. coli and to seedlings of Ginkgo biloba the cytoplasm and mitochondria; sterols,
Technische Universitt
Mnchen,
caused labelling patterns in ubiquinones sesquiterpenes and ubiquinones are formed
Lichtenbergstrasse 4, (S.T.J. Broers, PhD thesis, ETH Zrich, 1994) and predominantly via mevalonate. Isoprenoids
D-85747 Garching, ginkgolides (M.K. Schwarz, PhD thesis, ETH Zrich, synthesized in the plastids (hemiterpenes,
Germany.
1994) that were clearly inconsistent with the monoterpenes, diterpenes and carotenoids) are
*e-mail:
wolfgang.eisenreich@ mevalonate route. These labelling patterns suggested formed predominantly via 1-deoxyxylulose
ch.tum.de that C-3, C-2 and C-1 of a triose could supply C-1, C-2 5-phosphate (reviewed in Refs 46). The
http://plants.trends.com 1360-1385/01/$ see front matter 2001 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(00)01812-4
Review TRENDS in Plant Science Vol.6 No.2 February 2001 79
Fig. 1. Pathways of
isopentenyl diphosphate (a) (b)
(6) and dimethylallyl O
O O O O
diphosphate (7) 1 + 1
formation. The origins of + 9
SCoA SCoA 8 COO H O P O
carbon atoms from acetyl-
CoA (1), pyruvate (8) and OH O
glyceraldehyde 3-
phosphate (9) are CO2 dxs
indicated in blue, red and
green, respectively. (a) In O O OH O
the mevalonate pathway, 2 10
O
three molecules of acetyl-
SCoA O P O
CoA (1) are condensed
OH O
to 3-hydroxy-3- O
methylglutaryl-CoA (3) via 1 NADPH
dxr
acetoacetyl-CoA (2) by the SCoA
consecutive action of two
HSCoA NADP+
enzymes. Subsequent O
HO HO
reduction to mevalonate
(4), phosphorylation and 3 O P O 11
O
ATP-dependent HOOC COSCoA OH OH
decarboxylation produces
IPP (6), which can be 2 NADPH CTP
ispD NH2
converted to and from
DMAPP (7) by an 2 NADP+ HSCoA PPi
isomerase. (b) In
N
the deoxyxylulose HO HO O O
phosphate pathway, N O
D-glyceraldehyde
4 O P O P O O 12
3-phosphate (9) and HOOC CH2OH OH OH O O
pyruvate (8) are converted ATP
into 1-deoxy-D-xylulose HO OH
5-phosphate (10) in a ADP ATP ispE
decarboxylative reaction. ADP NH2
ATP
Subsequent
rearrangement and ADP O N
reduction leads to 2C-
HO O P O O
methyl-D-erythritol 4- O
N O
phosphate (11), which is
5
O O O O P O P O
converted into 2C-methyl-
O
D-erythritol 2,4-
HOOC CH2O P O P O OH OH O O
13
cyclodiphosphate (14) via O O
4-diphosphocytidyl-2C-
ispF HO OH
methyl-D-erythritol (12) ATP CMP
and 4-diphosphocytidyl- ADP + Pi CO2
2C-methyl-D-erythritol
2-phosphate (13). The O O
further reactions leading O O
O O P
to IPP (6) and DMAPP
6 P O
(7) are unknown. O P O P O O O
14
O O OH OH
O O O O
O O
7 O P O P O O P O
O P O P O O P
O O
O
O O O
6 7
compartmental separation of the two different IPP plants under physiological conditions (<1%). Higher
biosynthetic pathways is not absolute because at values have been found in plant cell cultures in the
least one metabolite can be exchanged between the presence of exogenous 1-deoxyxylulose or
compartments1113 (M.K. Schwarz, PhD thesis, ETH mevalonate. The nature of the metabolite(s)
Zrich, 1994). The extent of this crosstalk depends on exchanged between the compartments and the
the species as well as on the presence and regulation of the process remain to be established.
concentration of exogenous precursors. Generally, The crosstalk between the two terpenoid pathways
crosstalk contributions appear to be small in intact is one of the reasons for the historical failure to
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80 Review TRENDS in Plant Science Vol.6 No.2 February 2001
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Review TRENDS in Plant Science Vol.6 No.2 February 2001 81
identified that complemented mutants requiring be the binding site for NADPH (Refs 24,26,27).
2C-methyl-D-erythritol for growth23. The gene, now Fosmidomycin, a long-known antibiotic compound
designated dxr, was expressed in E. coli and the that mimics the structure of the hypothetical
recombinant protein shown to catalyse the formation reaction intermediate 2C-methyl-D-erythrose
of 2C-methyl-D-erythritol 4-phosphate from 1-deoxy- 4-phosphate, inhibits the reaction with a Ki of
D-xylulose 5-phosphate in a NADPH-dependent 38 nM (Refs 28,30).
reaction23 (Fig. 1).
The E. coli enzyme23,24 and its orthologues from the 4-Diphosphocytidyl-2C-methyl-D-erythritol synthase
bluegreen alga Synechocystis25, the plants Crude enzyme fractions from wild-type E. coli
Arabidopsis26 and M. piperita27, and the protist convert 2C-methyl-D-erythritol 4-phosphate into a
Plasmodium falciparum28 have been characterized in novel product in a CTP-dependent reaction31.
some detail. The E. coli enzyme was reported to have A database search for enzymes transferring a
a molecular mass of 140 kDa with four subunits. The cytidylphosphate residue to a polyol retrieved the
enzyme requires divalent metal ions such as Mn2+, asc1 gene of a serotype-specific DNA region from
Co2+ or Mg2+, and is strictly dependent on NADPH as Haemophilus influenzae. The N-terminal domain of
a cofactor23. The stereochemical features of the the cognate enzyme had been shown to catalyse the
reductoisomerase reaction have been studied with the formation of CDPribitol from ribitol 5-phosphate
recombinant enzymes from E. coli and Synechocystis, and CTP (Ref. 32). Subsequent similarity searches
showing that HSi from C-4 of NADPH is transferred to starting from that gene retrieved the hitherto
the HRe position at C-1 of 2C-methyl-D-erythritol unknown ygbP gene family (now designated ispD),
4-phosphate. Thus, 1-deoxy-D-xylulose 5-phosphate which is present in many bacteria as well as in
reductoisomerases from E. coli29 and Synechocystis25 Arabidopsis. More specifically, ispD is present in
are class B dehydrogenases. The kinetic properties of organisms with dxs and dxr genes but not in
the E. coli enzyme were also studied in some detail organisms devoid of dxs and dxr genes (Table 2).
(Table 1). Catalytic rates of 11.8 mol min1 mg1 were Thus, it appeared likely that ispD had a role in the
measured with Mn2+ at a pH optimum of 7.5. The novel terpenoid pathway.
enzyme does not accept 1-deoxy-D-xylulose as a This hypothesis was confirmed by studies on
substrate23. recombinant IspD protein, which was shown to
Site-directed mutagenesis of the recombinant catalyse the formation of 4-diphosphocytidyl-2C-
E. coli protein revealed that Glu231 is involved in the methyl-D-erythritol from 2C-methyl-D-erythritol
reaction and that His153, His209 and His257 might 4-phosphate and CTP (Refs 31,33; Fig. 1). Moreover, it
be involved in the binding of 1-deoxy-D-xylulose was shown that 4-diphosphocytidyl-2C-methyl-D-
5-phosphate24. The N-terminal region is thought to erythritol is incorporated efficiently into carotenoids
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82 Review TRENDS in Plant Science Vol.6 No.2 February 2001
Table 3. Putative ispD, ispE and ispF orthologues in incompletely sequenced organisms
Organism Gene
Eubacteria
Actinobacillus actinomycetes Contig320 Contig344 Contig320
Bacillus anthracis Banth_1680 Banth_1607 Banth_1680
Bacillus stearothermophilus Contig541 Contig541
Bordetella bronchisepticum Contig2244
Bordetella pertussis Contig301 Contig278 Contig301
Caulobacter crescentus C.cres._12574 C.cres._12574 C.cres._12574
Clostridium acetobutylicum C.aceto_gnl C.aceto_gnl C.aceto_gnl
Clostridium difficile Contig935 Contig985 Contig935
Corynebacterium diphteriae Contig239 Contig14 Contig239
Dehalococcoides ethenogenes Deth_1507 Deth_1545 Deth_1507
Desulfovibrio vulgaris Dvulg_gdv23 Dvulg_gdv23
Enterococcus faecalis Gef_10297 Gef_10286 Gef_10286
Geobacter sulfurreducens Gsulf_ggs98 Gsulf_ggs110 Gsulf_ggs98
Haemophilus ducreyi HTSC_730 HTSC_730 HTSC_730
Klebsiella pneumoniae Contig1719 Contig31 Contig1719
Legionella pneumophila CUGC_446 CUC_446
Mycobacterium avium M.avium_183 M.avium_24 M.avium_183
Mycobacterium bovis Contig442 Contig365 Contig353
Mycobacterium leprae Sanger_1769 Sanger_1769 Sanger_1769
Neisseria gonorrhoeae Contig9 Contig9 Contig9
Pasteurella multicoda CBCUMN_747 CBCUMN_747 CBCUMN_747
Porphyromonas gingivalis P.ging._GPG.con P.ging._GPG.con P.ging._GPG.con
Pseudomonas putida Pputida_10705 Pputida_10708 Pputida_10705
Rhodobacter capsulatus X12382 X12382
Salmonella enteritidis Contig1007
Salmonella paratyphi Spara_B_SPA.0.2916 Spara_B_SPA.0.2446 Spara_B_SPA.0.2916
Salmonella typhi S.typhi_CT18 S.typhi_CT18 S.typhi_CT18
Salmonella typhimurium Contig997 M77236 Contig997
Shewanella putrefaciens Sputr_6406 Sputr_6406 Sputr_6406
Streptomyces coelicor AL160331 AL359989 AL160331
Thiobacillus ferrooxidans T_ferro._6139 T_ferro._6154 T_ferro._6139
Treponema denticola Tdent_gtd269 Tdent_gtd51 Tdent_gtd269
Yersinia pestis Contig854 Contig855 Contig854
Zymomonas mobilis AF176314 AF088896 AF176314
Plants
Arabidopsis thaliana AF230737 AF288615 AC010852
Catharanthus roseus AF250236
Mentha piperita AF179283
Solanum esculentum AF263101
Protists
Plasmodium falciparum AF279661
indicates data not available.
of red pepper31 and that IspD is essential for IPP orthologous sets of dxs, dxr and ispD genes. Whole
biosynthesis in E. coli33. More recently, recombinant genome comparisons retrieved putative
IspD from Arabidopsis has been shown to catalyse the deoxyxylulose pathway genes from the completely
same reaction34. The kinetic properties of the E. coli sequenced genomes of organisms known to use the
and Arabidopsis enzyme are summarized in Table 1. deoxyxylulose pathway but not from the genomes of
Both enzymes require a divalent cation, preferably organisms that use the mevalonate pathway35. As a
Mg2+. The E. coli protein appears to be a dimer with hypothetical candidate following this distribution, the
25 kDa subunits31, but the Arabidopsis protein shows ychB gene with hitherto unknown function (now
a tendency for self-aggregation depending on the designated ispE) was identified in the genome of
buffer system34. Sequence analysis of the Arabidopsis E. coli and many other organisms35. The involvement
protein sequence revealed a putative plastid import of the orthologous gene product from tomato in
sequence34. chromoplast ripening had already been proposed36.
Both known plant orthologues (tomato and
4-Diphosphocytidyl-2C-methyl-D-erythritol kinase peppermint) carry putative plastid target
A bioinformatics approach has been used to identify sequences37,38. Sequence analysis revealed putative
more genes following the distribution of the ATP binding sites35,37,38.
http://plants.trends.com
Review TRENDS in Plant Science Vol.6 No.2 February 2001 83
The purified recombinant enzyme from E. coli was diphosphate (Fig. 1) is an intermediate of the
shown to catalyse the ATP-dependent deoxyxylulose phosphate pathway40.
phosphorylation of 4-diphosphocytidyl-2C-methyl-D-
erythritol into 4-diphosphocytidyl-2C-methyl-D- Further downstream reactions
erythritol 2-phosphate35,39 (Fig. 1, Table 1) at a rate35 The genes, enzymes and reactions involved in the
of 34 mol min1 mg1. The recombinant enzyme from conversion of 2C-methyl-D-erythritol 2,4-
tomato37 catalysed the same reaction at a rate of cyclodiphosphate into IPP and DMAPP are currently
33 mol min1 mg1 (Table 1). However, unknown. However, from a structural point of view, a
phosphorylation of isopentenyl monophosphate by ring-opening reaction, two dehydration steps and two
IspE from E. coli or M. piperita was reported, albeit at reduction steps are required to biosynthesize IPP and
extremely low rates at 1.0 nmol min1 mg1 and DMAPP. Moreover, there is evidence that IPP and
0.1 nmol min1 mg1, respectively38. More recent data DMAPP are biosynthesized via independent
with the recombinant enzymes from E. coli and mechanisms in the late steps of the novel pathway45,46
tomato37 gave catalytic rates below 0.002 nmol and that the gene product of lytB from E. coli is
min1 mg1. Therefore, it is hardly conceivable that involved in one of the late steps of IPP and DMAPP
phosphorylation of isopentenyl monophosphate biosynthesis47.
catalysed by IspE is metabolically relevant.
Possible applications
2C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase The rapidly increasing resistance of many human
The next step in the pathway was also identified pathogens to all currently available drugs generates
using bioinformatics. Genome analyses had shown an urgent demand for novel therapeutic approaches.
that many putative orthologues of the E. coli ygbB The deoxyxylulose phosphate pathway is used in
gene (now designated ispF) were linked or fused to many pathogenic microorganisms, as well as in
putative orthologues of ispD (Refs 31,40). Moreover, P. falciparum (Tables 2,3). In addition, the pathway
the occurrence of ispF correlates with the presence of does not occur in humans and animals. This makes
the deoxyxylulose pathway genes (Tables 2,3). Based the deoxyxylulose pathway of isoprenoid biosynthesis
on these findings, the E. coli ispF gene was expressed an ideal target for the development of novel
and the recombinant protein shown to catalyse the antibiotics and antimalarial agents. P. falciparum is
conversion of 4-diphosphocytidyl-2C-methyl-D- sensitive to the inhibition of this alternative
erythritol 2-phosphate into 2C-methyl-D-erythritol terpenoid biosynthetic pathway by fosmidomycin28.
Acknowledgements 2,4-cyclodiphosphate40,41 (Fig. 1). The enzyme In plants, the inhibition of the deoxyxylulose
Our work was
supported by grants
requires Mg2+ or Mn2+ but no other cofactors. pathway might be the basis for the development of
from the Deutsche The product, 2C-methyl-D-erythritol 2,4- novel herbicides19,48. Detailed knowledge of the
Forschungsgemeinschaft cyclodiphosphate, is well known as it accumulates mechanisms and regulation of the pathway will also
(SFB 369), the Fonds der
under oxidative stress in some bacteria4244. Results benefit the biotechnological production of
Chemischen Industrie and
the Hans-Fischer- obtained from incorporation experiments with commercially interesting isoprenoids, such as
Gesellschaft. chromoplasts of C. annuum suggest that the cyclic carotenoids49.
References 8 David, S. et al. (1981) 1-Deoxy-D-threo-2- thiamin, and pyridoxol. Proc. Natl. Acad. Sci.
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Pergamon heteroscyphic acid A in cell cultures of xylulose 5-phosphate synthase within the plastid
6 Rohmer, M. (1999) A mevalonate-independent Heteroscyphus planus: non-equivalent labelling of non-mevalonate pathway of isoprenoid
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2) (Cane, D., ed.), pp. 4568, Pergamon 14 Sprenger, G.A. et al. (1997) Identification of a mevalonate isoprenoid biosynthesis of plants as a
7 White, R.H. (1978) Stable isotope studies on the thiamin-dependent synthase in Escherichia coli test system for new herbicides and drugs against
biosynthesis of the thiazole moiety of thiamin in required for the formation of the 1-deoxy-D- pathogenic bacteria and the malaria parasite.
Escherichia coli. Biochemistry 17, 38333840 xylulose 5-phosphate precursor to isoprenoids, Z. Naturforsch. 55c, 305313
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20 Kuzuyama, T. et al. (2000) Cloning and Natl. Acad. Sci. U. S. A. 97, 64516456 42 Turner, D. et al. (1992) Structure determination of
characterization of 1-deoxy-D-xylulose 5-phosphate 35 Lttgen, H. et al. (2000) Biosynthesis of a novel cyclic phosphocompound isolated from
synthase from Streptomyces sp. strain CL190, which terpenoids: YchB protein of Escherichia coli Desulfovibrio desulfuricans. Biochem. J.
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J. Bacteriol. 182, 891897 Proc. Natl. Acad. Sci. U. S. A. 97, 10621067 to synthesize a new cyclodiphosphate correlates
21 Miller, B. et al. (1999) A Synechococcus 36 Lawrence, D.L. et al. (1997) Chromoplast with their tolerance to redox-cycling drugs: on a
leopoliensis SAUG 1402-1 operon harboring the development in ripening tomato fruit: crossroad of chemotherapy, environmental
1-deoxy-D-xylulose 5-phosphate gene and two identification of cDNAs for chromoplast-targeted toxicology and immunobiochemical problems.
additional open reading frames is functionally proteins and characterization of a cDNA encoding Biofactors 4, 6368
involved in the dimethylallyl diphosphate a plastid-localized low-molecular-weight heat 44 Ostrovsky, D. et al. (1998) Effect of oxidative
synthesis. FEBS Lett. 460, 485490 shock protein. Plant Mol. Biol. 33, 483492 stress on the biosynthesis of 2-C-methyl-D-
22 Lois, L.M. et al. (2000) Carotenoid biosynthesis 37 Rohdich, F. et al. (2000) Biosynthesis of erythritol-2,4-cyclopyrophosphate and
during tomato fruit development: regulatory role terpenoids: 4-diphosphocytidyl-2-C-methyl-D- isoprenoids by several bacterial strains. Arch.
of 1-deoxy-D-xylulose 5 phosphate synthase. Plant erythritol kinase from tomato. Proc. Natl. Acad. Microbiol. 171, 6972
J. 22, 503513 Sci. U. S. A. 97, 82518256 45 Giner, J-L. et al. (1998) Biosynthesis of
23 Takahashi, S. et al. (1998) A 1-deoxy-D-xylulose 38 Lange, M. and Croteau, R. (1999) Isopentenyl isoprenoids in Escherichia coli: the fate of the 3-H
5-phosphate reductoisomerase catalysing the diphosphate biosynthesis via a mevalonate- and 4-H atoms of 1-deoxy-D-xylulose. J. Chem.
formation of 2-C-methyl-D-erythritol 4-phosphate independent pathway: isopentenyl Soc., Chem. Commun. 18571858
in an alternative nonmevalonate pathway for monophosphate kinase catalyses the terminal 46 Rodriguez-Concepcion, M. et al. (2000) Genetic
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U. S. A. 95, 98799884 96, 1371413719 for the production of isopentenyl diphosphate and
24 Kuzuyama, T. et al. (2000) Characterization of 1- 39 Kuzuyama, T. et al. (2000) Studies on the dimethylallyl diphosphate in Escherichia coli.
deoxy-D-xylulose 5-phosphate reductoisomerase, an nonmevalonate pathway: conversion of FEBS Lett. 473, 328332
enzyme involved in isopentenyl diphosphate 4-(cytidine 5-diphospho)-2-C-methyl-D-erythritol 47 Cunningham, F.X., Jr et al. (2000) Evidence of a
biosynthesis, and identification of its catalytic amino to its 2-phospho derivative by 4-(cytidine role for LytB in the nonmevalonate pathway of
acid residues. J. Biol. Chem. 275, 1992819932 5-diphospho)-2-C-methyl-D-erythritol kinase. isoprenoid biosynthesis. J. Bacteriol. 182,
25 Proteau, P.J. et al. (1999) Stereochemistry of the Tetrahedron Lett. 41, 29252928 58415848
reduction step mediated by recombinant 1-deoxy- 40 Herz, S. et al. (2000) Biosynthesis of terpenoids: 48 Fellermeier, M. et al. (1999) Cell-free conversion of
D-xylulose 5-phosphate isomeroreductase. Org. YgbB protein converts 4-diphosphocytidyl-2C- 1-deoxy-D-xylulose 5-phosphate and 2-C-methyl-
Lett. 1, 921923 methyl-D-erythritol 2-phosphate to 2C-methyl-D- D-erythritol 4-phosphate into -carotene in higher
26 Schwender, J. et al. (1999) Cloning and erythritol 2,4-cyclodiphosphate. Proc. Natl. Acad. plants and its inhibition by fosmidomycin.
heterologous expression of a cDNA encoding 1- Sci. U. S. A. 97, 24862490 Tetrahedron Lett. 40, 27432746
deoxy-D-xylulose-5-phosphate reductoisomerase 41 Takagi, M. et al. (2000) Studies on the 49 Matthews, P.D. and Wurtzel, E.T. (2000) Metabolic
of Arabidopsis thaliana. FEBS Lett. 455, 140144 nonmevalonate pathway: formation of 2-C- engineering of carotenoid accumulation in E. coli
27 Lange, B.M. and Croteau, R. (1999) Isoprenoid methyl-D-erythritol 2,4-cyclodiphosphate from by modulation of the isoprenoid precursor pool
biosynthesis via a mevalonate-independent 2-phospho-4-(cytidine 5-diphospho)-2-C-methyl- with expression of deoxyxylulose phosphate
pathway in plants: cloning and heterologous D-erythritol. Tetrahedron Lett. 41, 33953398 synthase. Appl. Microbiol. Biotechnol. 53, 396400
expression of 1-deoxy-D-xylulose-5-phosphate
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mevalonate pathway of isoprenoid biosynthesis as
antimalarial drugs. Science 285, 15731576
Trends in Plant Science online
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Chapter 24
Lipid Biosynthesis
........................
Chapter Outline
Fatty acid biosynthesis
Biosynthesis localized in cytosol: Fatty acid degradation in mitochondria
Intermediates held on acyl carrier protein (ACP): Phosphopantetheine group attached to
serine: CoA in degradation
Fatty acid synthase: Multienzyme complex
Carbons derived from acetyl units
Acetyl CoA to malonyl CoA by carboxylation
Acetyl unit added to fatty acid with decarboxylation of malonyl CoA
Carbonyl carbons of acetyl units reduced using NADPH
Source of acetyl units
Amino acids, glucose
Acetyl CoA used to produce citrate
Citrate exported to cytosol: ATP-citrate lyase forms acetyl-CoA and oxaloacetate
Source of NADPH
Oxaloacetate utilization
Oxaloacetate (from citrate) to malate: NADH dependent reaction
Malate to pyruvate: Malic enzyme: NADPH produced
Pentose phosphate pathway
Malonyl-CoA production: Acetyl-CoA carboxylase
Biotin-dependent enzyme
ATP drives carboxylation
Enzyme regulation
Filamentous polymeric form active
Citrate favors active polymer
Palmitoyl-CoA favors inactive protomer (polymers monomer or
building block molecule)
Citrate/palmitoyl-CoA effects depend on state of phosphorylation of
protein
o Unphosphorylated protein binds citrate with high affinity:
Activation
o Phosphorylated protein binds palmitoyl with high affinity:
Inactivation
Acetyl transacetylase: Acetylates acyl carrier protein (ACP): Destined to become methyl end of
fatty acid
Malonyl transacetylase: Malonylates ACP
-Ketoacyl-ACP synthase (acyl-malonyl ACP condensing enzyme): Accepts acetyl group:
Transfers acyl group to malonyl-ACP
Malonyl carboxyl group released: Decarboxylation drives synthesis
Malonyl-ACP converted to acetoacetyl-ACP
-Ketoacyl-ACP reductase
Carbonyl carbon reduced to alcohol
Chapter 24 Lipid Biosynthesis
385
Chapter 24 Lipid Biosynthesis
DHAP acetylated
Acyl group exchanged for alcohol
Keto group on DHAP reduced to alcohol and acylated
Head group attached
Desaturase produces double bonds
Sphingolipid biosynthesis
Serine and palmitoyl-CoA condensed with decarboxylation to produce 3-
ketosphinganine
Reduction forms sphingamine
Sphingamine acylated to form N-acyl sphingamine
Desaturase produces ceramide
Cerebrosides: Galactose or glucose added
Gangliosides: Sugar polymers: Sugars derive from UDP-monosaccharides
Eicosanoids: Derived from 20-C fatty acids: Arachidonate is precursor
Local hormones: Prostaglandins, thromboxanes, leukotrienes, hydroxyeicosanoic acids
Prostaglandins
Cyclopentanoic acid formed from arachidonate by prostaglandin
endoperoxidase synthase
Aspirin inhibits enzyme
Cholesterol
Membrane component
Precursor of important biomolecules
Bile salts
Steroid hormones
Vitamin D
Cholesterol biosynthesis: In liver
Mevalonate biosynthesis
Thiolase condenses two acetyl-CoA to produce acetoacetyl-CoA
HMG-CoA synthase produces HMG-CoA
HMG-CoA reductase produces mevalonate
Rate limiting step
Regulation
o Inactivated by cAMP-dependent protein kinase
o Short half life of enzyme when cholesterol levels high
o Gene expression regulated
Pharmacological target for blood cholesterol regulation
Isopentenyl pyrophosphate and dimethylallyl pyrophosphate from mevalonate
Squalene to lanosterol to cholesterol
Lipid transport
Fatty acids complexed to serum albumin
Phospholipids, triacylglycerol, cholesterol transported as lipoprotein complexes
Lipoprotein complex types: HDL, LDL, IDL, VLDL, Chylomicrons
Chylomicrons formed in intestine
HDL, VLDL assembled in liver
o Core of triacylglycerol
o Single layer of phospholipid
o Proteins and cholesterol inserted
o VLDL to IDL to LDL to liver for uptake and degradation
o HDL: Assembled without cholesterol but picks up cholesterol
during circulation
Bile salts
Glycocholic acid
Taurocholic acid
Steroid hormones
Cholesterol to pregnenolone
Pregnenolone to progesterone
Hormone
386
Chapter 24 Lipid Biosynthesis
Complex Lipids
The glycerolipids, including glycerophospholipids and triacylglycerols, are synthesized from
glycerol, fatty acids, and head groups. Synthesis starts with the formation of phosphatidic acid
from glycerol-3-phosphate and fatty acyl-CoA. C-1 is esterified usually with a saturated fatty
acid. Phosphatidic acid may be converted to diacylglycerol and then to triacylglycerol.
Alternately, diacylglycerol can be used to synthesize phosphatidylethanolamine and
phosphatidylcholine with CDP-derivatized head groups serving as substrates. Phosphatidylserine
is produced by exchange of the ethanol head-group from PE with serine. Phosphatidylinositol,
phosphatidylglycerol, and cardiolipin (two diacylglycerols linked together by glycerol) are
synthesized using CDP-diacylglycerol as an intermediate. Plasmalogens are synthesized from
acylated DHAP. The acyl group is exchanged for a long-chain alcohol followed by reduction of the
keto carbon of DHAP, acyl group transfer from acyl-CoA to C-2, head group transfer from CDP-
ethanolamine and formation of a cis double bond between C-1 and C-2 of the long-chain alcohol.
The sphingolipids all derive from ceramide, whose synthesis starts with bond formation
between palmitic acid and the -carbon of serine (with loss of the serine carboxyl carbon as
bicarbonate). After a few steps a second fatty acid is attached to serine in amide linkage.
Subsequent sugar additions lead to cerebrosides and gangliosides.
Prostaglandins
387
Chapter 24 Lipid Biosynthesis
The prostaglandins are produced from arachidonic acid released by phospholipase A2 action
on phospholipids. Production of these local hormones is blocked by aspirin, and nonsteroid anti-
inflammatory agents such as ibuprofen and phenylbutazone.
Cholesterol
Cholesterol derives from HMG-CoA, a product we already encountered in ketone body
formation. You might recall that ketone bodies are produced from acetyl-CoA units. HMG-CoA is
a six-carbon CoA derivative produced from three acetyl units. The rate-limiting step in
cholesterol synthesis is formation of 3R-mevalonate from HMG-CoA by HMG-CoA reductase,
which catalyzes two NADPH-dependent reductions. This enzyme is carefully regulated by 1)
phosphorylation leading to inactivation, 2) degradation, and 3) gene expression. Mevalonate, a
six-carbon intermediate, is converted to isopentenyl pyrophosphate, which is used to synthesize
cholesterol. Cholesterol is the precursor of bile salts and the steroid hormones. You should
understand how lipoproteins are responsible for movement of cholesterol and other lipids in the
body.
388
Chapter 24 Lipid Biosynthesis
Figure 24.7 The pathway of palmitate synthesis from acetyl-CoA and malonyl-CoA. Acetyl and malonyl
building blocks are introduced as acyl carrier protein conjugates. Decarboxylation drives the -ketoacyl-ACP
synthase and results in the addition of two-carbon units to the growing chain. Concentrations of free fatty
acids are extremely low in most cells, and newly synthesized fatty acids exist primarily as acyl-CoA esters.
389
Chapter 24 Lipid Biosynthesis
2. Use the relationships shown in Figure 24.1 to determine which carbons of glucose will
be incorporated into palmitic acid. Consider the cases of both citrate that is immediately
exported to the cytosol following its synthesis and citrate that enters the TCA cycle.
Answer: The six carbons of glucose are converted into two molecules each of CO2 and acetyl
units of acetyl-coenzyme A. Carbons 1, 2, and 3 of glyceraldehyde derive from carbons 3 and 4, 2
and 5, and 1 and 6 of glucose respectively. Carbon 1 of glyceraldehyde is lost as CO2 in
conversion to acetyl-CoA, so we expect no label in palmitic acid from glucose labeled only at
carbons 3 and 4. The carbonyl carbon and the methyl carbon of the acetyl group of acetyl-CoA
derive from carbons 2 and 5, and carbons 1 and 6 of glucose, respectively. The methyl carbon is
incorporated into palmitoyl-CoA at every even-numbered carbon, whereas the carbonyl carbon is
incorporated at every odd-numbered carbon.
Acetyl-CoA is produced in the mitochondria and exported to the cytosol for fatty acid
biosynthesis by being converted to citrate. The cytosolic enzyme, citrate lyase, converts citrate to
acetyl-CoA and oxaloacetate. When newly synthesized citrate is immediately exported to the
cytosol, the labeling pattern described above will result. However, where citrate is instead
metabolized in the citric acid cycle, back to oxaloacetate, label derived from acetyl-CoA shows up
at carbons 1, 2, 3 and 4 of oxaloacetate. These carbons do not get incorporated into palmitoyl-
CoA.
3. Based on the information presented in the text and in Figures 24.4 and 24.5, suggest
a model for the regulation of acetyl-CoA carboxylase. Consider the possible roles of
subunit interaction, phosphorylation, and conformation changes in your model.
Answer: Acetyl-CoA carboxylase catalyzes the formation of malonyl-CoA, the committed step in
synthesis of fatty acids. This enzyme is a polymeric protein composed of protomers, or subunits,
of 230 kD. In the polymeric form, the enzyme is active whereas in the protomeric form the
enzyme is inactive. Polymerization is regulated by citrate and palmitoyl-CoA such that citrate, a
metabolic signal for excess acetyl units, favors the polymeric and, therefore, active form of the
enzyme whereas palmitoyl-CoA shifts the equilibrium to the inactive form. The activity of acetyl-
CoA carboxylase is also under hormonal regulation. Glucagon and epinephrine stimulate cyclic
AMP-dependent protein kinase that will phosphorylate a large number of sites on the enzyme.
The phosphorylated form of the enzyme binds citrate poorly and citrate binding occurs only at
high citrate levels. Citrate is a tricarboxylic acid with three negative charges and its binding site
on the enzyme is likely to be composed of positively-charged residues. Phosphorylation
introduces negative charges, which may be responsible for the decrease in citrate binding.
In the phosphorylated form, low levels of palmitoyl-CoA will inhibit the enzyme. Thus, the
enzyme is sensitive to palmitoyl-CoA binding and to depolymerization in the phosphorylated form.
If we assume that the palmitoyl-CoA binding site is located at a subunit-subunit interface, and
390
Chapter 24 Lipid Biosynthesis
that phosphorylated, and hence negatively charged subunits interact with lower affinity than do
unphosphorylated subunits, we see that it is easier for palmitoyl-CoA to bind to the enzyme.
4. Consider the role of the pantothenic acid groups in animal fatty acyl synthase and the
size of the pantothenic acid group itself, and estimate a maximal separation between the
malonyl transferase and the ketoacyl-ACP synthase active sites.
Answer: In fatty acyl synthase, pantothenic acid is attached to a serine residue as shown below.
O O CH3 O
H
HS CH2 C CH2 C C P serine
CH2 N CH2 N C O OH
CH3 OH
H H OH
The approximate distance from the pantothenic group to the -carbon of serine is calculated as
follows. For carbon-carbon single bonds the bond length is approximately 0.15 nm. The distance
between carbon atoms is calculated as follows.
109o
C
0.15 nm
35.5 o 35.5 o
C
d
d = 0.15nm cos(35.5 ) = 0.22nm
Let us use this length for carbon-carbon single bonds, carbon-oxygen bonds, oxygen-
phosphorous bonds, and carbon-nitrogen bonds exclusive of the amide bond. For the amide
bond we will use a distance of 0.132 nm. The overall length is approximately 1.85 nm from the -
carbon of serine to the sulfur. The maximal separation between malonyl transferase and
ketoacyl-ACP is about twice this distance or approximately 3.7 nm. The actual distance between
these sites is smaller than this upper limit.
5. Carefully study the reaction mechanism for the stearoyl-CoA desaturase in Figure
24.14, and account for all of the electrons flowing through the reactions shown. Also
account for all of the hydrogen and oxygen atoms involved in this reaction, and convince
yourself that the stoichiometry is correct as shown.
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Chapter 24 Lipid Biosynthesis
7. Write a balanced, stoichiometric reaction for the synthesis of cholesterol from acetyl-
CoA.
3 ATP 3 ADP +
2 NADPH 2 NADP+ + H2O Pi + CO2
OH O OH
-OOC CH2 C CH2 C S-CoA -
OOC CH2 C CH2 CH2 OH
CH3 CoA-SH CH3
HMG-CoA mevalonate
CH3 O O CH3 O O
CH2 C CH2 CH2 O P O P OH CH3 C CH CH2 O P O P OH
OH OH OH OH
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Chapter 24 Lipid Biosynthesis
O- O-
O- P O P O
O O
+ O O
O P O P O-
O- O-
NADPH
NADP+
+ 2 PPi
squalene
Squalene is converted to lanosterol in two steps catalyzed by squalene epoxidase and squalene
oxidocyclase. Squalene + 0.5 O2 + NADPH lanosterol
The overall equation for acetyl-CoA to lanosterol is:
18 Acetyl-CoA + 13 NADPH +13 H+ + 18 ATP + 0.5 O2
Lanosterol + 18 CoA-SH + 13 NADP+ + 18 ADP + 6 Pi + 6 PPi + 6 CO2
The pathway from lanosterol to cholesterol involves the oxidation and loss of three carbons.
8. Trace each of the carbon atoms of mevalonate through the synthesis of cholesterol,
and determine the source (i.e., the position in the mevalonate structure) of each carbon in
the final structure.
Answer:
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Chapter 24 Lipid Biosynthesis
mevalonate
OH
-
OOC 4C 3C 2C 1C OH
4C
4C O O 4C O O
4C 3C 2C 1C O P O P O- 4C 3C 2C 1C O P O P O-
O- O- O- O-
O- P O P O 2C 4C 4C
1C 3C
4C O + O
O O 4C
3C 1C
4C 4C 2C O P O P O-
3C 1C O- O-
4C 4C 2C
NADPH
3C 1C
4C 2C
farnesyl pyrophosphate 2C 4C
1C 3C
+
NADP
2C 4C 4C
+ 2 PPi
1C 3C
4C
2C 4C 4C
3C 1C
2C 1C 3C
4C 4C
4C
3C 1C squalene
4C 4C 2C
3C 1C
4C 2C
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Chapter 24 Lipid Biosynthesis
2C 4C
1C 3C
2C 4C 4C
1C 3C
4C
2C 4C 4C
3C 1C
2C 1C 3C
4C 4C
4C
3C 1C squalene
4C 4C 2C
3C 1C
4C 2C 4C
4C
3C
4C
2C 4C
3C
1C
2C
1C 1C
1C 2C
3C 4C
4C 2C
4C
1C 3C 4C
3C
4C
4C
2C 2C
1C
3C
4C 4C 4C 2C 4C
4C
3C 1C 3C
4C
1C 4C
2C
1C 2C
4C 1C
4C 2C 3C
4C
1C 3C 3C
2C 2C 4C
HO 3C 1C
cholesterol
9. Suggest a structural or functional role for the O-linked saccharide domain in the LDL
receptor (Figure 24.40).
Answer: LDL receptors are synthesized on the rough endoplasmic reticulum and move through
the smooth endoplasmic reticulum and Golgi apparatus before being incorporated into the
plasma membrane. On the plasma membrane, LDL receptors bind LDL, aggregate into patches,
and are internalized into coated vesicles that fuse with lysosomes where LDL is degraded. The O-
linked saccharide domain functions to extend the receptor domain away from the cell surface,
above the glycocalyx coat. This allows the receptor to bind circulating lipoproteins.
10. Identify the lipid synthetic pathways that would be affected by abnormally low levels
of CTP.
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Chapter 24 Lipid Biosynthesis
11. Determine the number of ATP equivalents needed to form palmitic acid from acetyl-
CoA. (Assume for this calculation that each NADPH is worth 3.5 ATP.)
Answer:
O
H3C (CH2)14 C S CoA
Palmitoyl-CoA enzyme
enzyme HB
:B
H
HO CH2 C- COO-
HO CH2 C COO-
+
+ H NH
H NH
C C
OH OH
2-
2-
O3PO O3PO
N+ CH3 N+ CH3
Seryl-pyridoxal phosphate
H3C
H 3C
CoASH
(CH2)14
(CH2)14
C O
C O
HO CH2 C enzyme O-
HB HO CH2 C C
+
H NH
+ NH O
C- H
C
OH
2-
O3PO OH
2-
O3PO
N+ CH3
N+ CH3
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Chapter 24 Lipid Biosynthesis
H 3C
H 3C (CH2)14
(CH2)14 C O
H 2O HO CH2 C H
C O
NH3+
HO CH2 C H
+
H NH H O
C C
OH OH
2- 2-O PO
O3PO 3
N+ CH3 N+ CH3
13. Why is the involvement of FAD important in the conversion of stearic acid to oleic
acid?
14. Write a suitable mechanism for the HMG-CoA synthase reaction. What is the
chemistry that drives this condensation reaction.
Answer: The mechanism for HMG-CoA was already discussed in problem 14 of chapter 23. The
chemistry is not unlike that of citrate synthase. HMG-CoA synthase produces
hydroxymethylglutaryl-CoA from acetyl-CoA and acetoacetyl-CoA. The reaction is accompanied
by hydrolysis of a thioester bond linking coenzyme to the acetyl group. We learned in earlier
chapters that these thioester bonds are high energy. Production of fatty acyl-CoA by acyl-CoA
synthetase, which is used to activate fatty acids for beta oxidation, uses in effect two
phosphoanhydride bonds to create the thioester bond. (As an interesting aside, synthase and
synthetase both run reactions that join two substrates to form a product. Synthetases, in
general, drive these reaction with hydrolysis of high-energy phosphoanhydride bonds. Synthases
do not.)
15. Write a suitable reaction mechanism for the -ketoacyl ACP synthase, showing how
the involvement of malonyl-CoA drives this reaction forward.
Answer: -Ketoacyl ACP synthase links an acetyl group from malonyl-ACP onto an acyl-group
(acetyl- in the first round) during fatty acid synthesis. Malonyl-CoA is, in effect, an activated
acetyl group produced at the expense of hydrolysis of ATP by acetyl-CoA carboxylase. The
mechanism of action involves decarboxylation of malonyl-CoA to produce a carbanion on the beta
carbon, which attacks the carbonyl carbon of acyl-ACP producing -ketoacyl ACP. This
mechanism is shown below.
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Chapter 24 Lipid Biosynthesis
-O O CO2 O O O
16. Consider the synthesis of linoleic acid from palmitic acid and identify a series of
three consecutive reactions that embody chemistry similar to three reactions in the
tricarboxylic acid cycle.
Answer: Palmitic acid is a saturated 16-carbon fatty acid whereas linoleic acid shown below- is
18 carbons long with two carbon-carbon double bonds. To convert palmitic acid to linoleic acid it
must first be elongated using one cycle of fatty acid synthesis. Elongation of palmitoyl-CoA
involves a thiolase reaction using acetyl-CoA, which adds to the carboxyl end. The -keto acyl
CoA derivative is then reduced to -hydroxy, dehydrated to form a carbon-carbon double bond
and then reduced to produce stearyl-CoA. The chemistry of this series of reactions is similar to
the chemistry found in the TCA cycle but going in reverse from oxaloacetate to succinate.
C
H3C OH
17. Rewrite the equation in Section 24.1 to describe the synthesis of behenic acid (see
Table 8.1).
Answer: Behenic acid (a.k.a., docosanoic acid) is a 22-carbon long fatty acid shown below. On
page 770 we are given the equation for synthesis of palmitoyl-CoA, a 16-carbon long fatty acid.
To produce behenic acid we need to recognize that we will need to run three more cycles of fatty
acid synthesis. Each cycle will consume one acetyl-CoA, 1 ATP and 2 NADPH.
H3C COOH
The equation is:
11-Acetyl-CoA + 10 ATP4- + 20 NADPH + 10 H+ J behenoyl-CoA + 20 NADP+ + 10 CoA-SH + 10
ADP3- + 10 Pi2-. (There are only 10 H+ consumed, and not 20 (thinking that each NADPH used is
actually used with a proton), because hydrolysis of ATP releases a proton. You can see this by
counting charge in the equation.)
2. Although acetate units are incorporated into fatty acids during synthesis, they derive from
three-carbon compounds attached to coenzyme A. What is this three-carbon coenzyme A
derivative? What enzyme is responsible for its formation? How many high-energy phosphate
bonds are cleaved to drive its synthesis?
3. Describe how, in animals, the activity of acetyl-CoA carboxylase is regulated by citrate and
palmitoyl-CoA and how this regulation is sensitive to covalent modification of the enzyme.
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Chapter 24 Lipid Biosynthesis
5. From the following list of compounds identify those that are cholesterol derivatives and
appropriately identify each cholesterol derivative as a hormone (H), bile salt (B), or vitamin (V).
a. Prostaglandin D2
b. Glycocholic acid.
c. Squalene
d. Testosterone
e. Arachidonic Acid.
f. Cortisol
g. Cholecalciferol
h. Progesterone
i. Thromboxanes
j. Taurocholic acid
k. Aldosterone
8. In eukaryotes, glycerolipids are all derived from phosphatidic acid. Draw the structure of
phosphatidic acid and outline its biosynthesis from dihydroxyacetone-phosphate and from
glycerol-3-phosphate.
10. Fill in the blanks. The prostaglandins are that function locally and at very low
concentrations. They are synthesized from , a 20-carbon polyunsaturated fatty acid.
Mammals can produce this fatty acid from (18:29,12) but must acquire this polyunsaturated
fatty acid from their diet.
Answers
1. Acetate units on acetyl-CoA are used to produce citrate in the mitochondria. Citrate is
exported to the cytosol where it is converted to acetyl-CoA and oxaloacetate. Citrate inhibits
phosphofructokinase and thus serves as a regulator of glycolysis. It also stimulates fatty acid
synthesis.
3. Acetyl-CoA carboxylase is active in a polymeric state. The equilibrium between active polymer
and inactive protomers is affected by citrate and palmitoyl-CoA. Citrate is an allosteric activator
of the enzyme and shifts the equilibrium to the polymer. Palmitoyl-CoA shifts the equilibrium to
the inactive, protomeric state. The enzyme is phosphorylated by a number of kinases and the
phosphorylated state has a low affinity for citrate and a high affinity for palmitate.
4. a. 3; b. 6; c. 2; d. 4; e. 1; f. 5.
5. b. B; d. H; f. H; g. V; h. H; j. B; k. H.
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Chapter 24 Lipid Biosynthesis
7. a. 2; b. 4; c. 1; d. 3.
O
R2 C O C H O
CH2 O C O-
O-
9. The head groups of phosphatidylethanolamine and phosphatidylcholine derive from cytidine
diphosphate derivatives. CDP-diacylglycerol is a precursor of phosphatidylinositol,
phosphatidylglycerol, and cardiolipin.
Additional Problems
1. What are the sources of carbons for fatty acid biosynthesis? What is the role of the citrate-
malate-pyruvate shuttle in making carbon compounds available for fatty acid biosynthesis?
2. Movement of citrate out of the mitochondria coordinates glycolysis and fatty acid biosynthesis.
Explain.
3. Name the three water soluble vitamins that are crucial to fatty acid synthesis and briefly
describe the roles they play in this process.
6. Lovastatin lowers serum cholesterol by interfering with HMG-CoA reductase, the enzyme that
catalyzes the rate limiting step in cholesterol synthesis. The drug is administered as an inactive
lactone that is activated by hydrolysis to mevinolinic acid, a competitive inhibitor of HMG-CoA
reductase. Can you recall another lactone hydrolysis reaction encountered in an earlier chapter?
7. What is the role of high-density lipoproteins in regulation of cholesterol levels in the blood?
8. Synthesis of the steroid hormones from cholesterol starts with the reaction catalyzed by
desmolase shown below. Why is this a critical reaction for formation of steroid hormones?
400
Chapter 24 Lipid Biosynthesis
H3C
H3C
O
HO
Cholesterol C H
Isocaproic aldehyde
O
H3C
H3C
HO
Pregnenolone
Abbreviated Answers
1. The immediate source of carbons is acetyl-CoAs, which are produced from carbohydrates,
amino acids, and lipids. Acetyl-CoA is produced in the mitochondria but fatty acid biosynthesis
occurs in the cytosol. To move acetyl units out of the mitochondria, they are condensed onto
oxaloacetate to form citrate, in a citric acid cycle reaction. Citrate is then transported out of the
mitochondria to the cytosol, where ATP-citrate lyase catabolizes citrate to acetyl-CoA and
oxaloacetate. This cytosolic acetyl-CoA is used to synthesize fatty acids. So, the citrate-malate-
pyruvate shuttle is responsible for moving two-carbon units from the mitochondria to the cytosol.
However, it has another purpose: it supplies some of the NADPH needed for fatty acid synthesis.
Cytosolic oxaloacetate is reduced to malate and then oxidatively decarboxylated to CO2 and
pyruvate by malic enzyme, in an NADP+-dependent reaction. The NADPH thus formed is
consumed during the reduction steps of fatty acid biosynthesis.
2. When glycolysis was covered, it was pointed out that phosphofructokinase activity is
inhibited by citrate. In this chapter, we saw how citrate is used to move two-carbon units from
the mitochondria to the cytosol for fatty acid biosynthesis. An increase in the concentration of
citrate is a signal that the citric acid cycle is backing up, either because energy stores are
satisfactory or because there is an abundance of acetyl units. In either case, there is not reason
to continue glycolysis. Movement of citrate out of the mitochondria shifts acetyl units from
degradation via the citric acid to storage via cytosolic fatty acid synthesis and serves to turn down
glycolysis at phosphofructokinase.
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Chapter 24 Lipid Biosynthesis
4. Mammals cannot introduce a double bond beyond C-9 in a given fatty acid. The
prostaglandins are synthesized from linoleic acid, 9,12-octadecadienoic acid, which cannot be
produced by mammals and is therefore an essential fatty acid.
5. The components of glycerophospholipids are glycerol, phosphate, fatty acids, and an alcoholic
head group. Synthesis starts with either glycerol (via reduction of glyceraldehyde) or DHAP being
converted to glycerol-3-phosphate by glycerokinase or glycerol-3-phosphate dehydrogenase,
respectively. Glycerol-3-phosphate is converted to 1-acylglycerol-3-P and then to phosphatidic
acid (1,2-diacylglycerol-3-P) by two acyltransferase reactions. Phosphatidic acid serves as the
precursor for triacylglycerol and the glycerophospholipids phosphatidylethanolamine (PE),
phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol
(PG), and cardiolipin (diphosphatidylglycerol). Phosphatidic acid is converted to diacylglycerol,
which is converted to PE or PC by transferases using CDP-derivatized ethanolamine or choline.
Alternatively, phosphatidic acid can be converted to its CDP derivative, CDP-diacylglycerol, which
is metabolized to PI or PG. PS is produced from PE using serine to displace ethanolamine.
7. HDL is assembled in the endoplasmic reticulum of liver cells and secreted into the blood.
Newly synthesized HDL contains very little cholesterol but with time it accumulates cholesterol as
both free cholesterol and as cholesterol esters. HDL then returns to the liver where cholesterol is
either stored or converted to bile salts and excreted?
8. The steroid hormones are transported in the blood to target tissues and must therefore be
slightly more soluble than cholesterol. The reaction catalyzed by desmolase removes the
hydrocarbon tail of cholesterol, making the product more soluble.
Summary
The biosynthesis of lipid molecules proceeds via mechanisms and pathways, which are
different from those of their degradation. In the synthesis of fatty acids, for example, 1)
intermediates are linked covalently to the -SH groups of acyl carrier proteins instead of coenzyme
A, 2) synthesis occurs in the cytosol instead of the mitochondria, 3) the nicotinamide coenzyme
used is NADPH instead of NADH, and 4) in eukaryotes, the enzymes of fatty acid synthesis are
associated in one large polypeptide chain instead of being separate enzymes. Fatty acids are
synthesized by the addition of two carbon acetate units, which have been activated by the
formation of malonyl-CoA, decarboxylation of which drives the reaction forward. Once the
growing fatty acid chain reaches 16 carbons in length, it dissociates from the fatty acid synthase
and is subject to the introduction of unsaturations or additional elongation. Acetyl-CoA needed
for fatty acid synthesis is provided in the cytosol by citrate that is transported across the
mitochondrial membrane and converted to acetyl-CoA and oxaloacetate by ATP-citrate lyase.
Formation of malonyl-CoA by acetyl-CoA carboxylase (ACC), a biotin-dependent enzyme, commits
acetate units to fatty acid synthesis. In animals, ACC is a multifunctional protein, which forms
long, filamentous polymers. It is allosterically activated (and polymerized) by citrate and
inhibited (and depolymerized) by palmitoyl-CoA. Affinities for both these regulators are decreased
by phosphorylation of the enzyme at up to 8 to 10 separate sites. The fatty acid synthesis
reactions involve formation of O-acetyl and O-malonyl enzyme intermediates, followed by transfer
of the acetyl group to the -SH of an acyl carrier protein (ACP) and then to the -ketoacyl-ACP
synthase. Transfer of the malonyl group to the ACP is followed by decarboxylation of the malonyl
group and condensation of the remaining two-carbon unit with the carbonyl carbon of the acetate
group on the synthase. This is followed by reduction of the -carbonyl to an alcohol, dehydration
to yield a trans-, double bond and reduction to yield a saturated bond. Introduction of
unsaturations in the nascent chain occurs by O2-dependent and O2-independent pathways and
may be followed by further chain elongation. Several mechanisms are utilized to introduce
multiple unsaturations in a fatty acid chain. Regulation of fatty acid synthesis is related to
regulation of fatty acid breakdown and the activity of the TCA cycle, because of the importance of
acetyl-CoA in all these processes. Malonyl-CoA inhibits carnitine transport, blocking fatty acid
oxidation. Citrate activates ACC and palmitoyl-CoA inhibits, both in chain-length-dependent
fashion. The enzymes of fatty acid synthesis are also under hormonal control.
Glycerolipid synthesis is built around the synthesis of phosphatidic acid from glycerol-3-
phosphate or dihydroxyacetone phosphate. Specific acyltransferases add acyl chains to these
glycerol derivatives. Other glycerolipids, such as phosphatidylcholine (PC) and
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Chapter 24 Lipid Biosynthesis
403
16
1. Introduction
Plants synthesize an astonishing diversity of isoprenoids, some of which play essential roles
in photosynthesis, respiration, and the regulation of growth and development. In spite of
economic significance of the terpenoids and their many essential functions, relatively little is
known about terpenoid metabolism and its regulation in plants (Mansouri et al, 2009).
However, two independent patheways for the biosynthesis of isoprenoid precursors coexist
within the plant cell: the cytosolic mevalonic acid (MVA) pathway and the plastidial
methyerythritol phosphate (MEP) pathway. Cannabis is a diecious species that is a source of
fiber, food, oil and medicine. Cannabinoids represent a distinctive class of compounds
belong to the chemical class of natural terpenophenols. Among others, 9-tetrahycannabinol
(THC) and cannabidiol (CBD) are the most important of these compounds. The experiments
with labeling patterns showed that the cannabinoids are derived entirely or predominantly
(98%) from the deoxyxylulose pathway (Fellemeier et al., 2001). They are produced by
glandular trichomes that occur on most aerial surfaces of the plant (Hilling 2004). Cannabis
is used in modern medicine for the treatment of emesis in chemotherapy. As well as being
useful anti-emetics, cannabinoids appear to have therapeutic value as antispasmodics,
analgesics, and appetite stimulants and have potential in the treatment of epilepsy,
glaucoma, and asthma (Agurell and Nilsson 1972; Guzman 2003; Howlett et al., 2004).
However little is known about the effects of plant hormones on the regulation of these
pathways. Thus, we investigated the effect of gibberellic acid (GA3) on changes in the amount
of many produced terpenoids and the activity of the key enzymes, 1-deoxy-D-xylulose 5-
phosphate synthase (DXS) and 3-hydroxy-3-methy glutaryl coenzyme A reductase (HMGR) in
these pathways. To understand the role of gibberellic acid (GA3) in the regulation of two
terpenoid biosynthesis pathways, we gained experience on the response of the main end
products of these pathways and cannabinoids under GA3 treatment in cannabis plants.
This chapter will provide focus on recent development of the interaction effects of
terpenoids and gibberellic acid in Cannabis sativa.
2. Biosynthesis of terpenoids
Isoprenoids (also known as terpenoides) made from assembly of isoprene units. Isoprenes are
flexible five carbon units (CH2)2C=CH-CH2- , and they comprise a diverse class of plant
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346 Advances in Selected Plant Physiology Aspects
metabolites; most of them are known as defense-related compounds, flavors or scents (Chris et
al., 2007). Terpenoids constitute the largest family of natural plant products, with over 30,000
members (Dewick, 2002). Members of this diverse group of natural products are found in all
organisms. In primary metabolism, they function as photosynthetic pigments (chlorophylls
and carotenoids), electron transport (ubiquinone and plastoquinone), plant hormones (abscisic
acid and gibberellins) and membrane fluidity such as sterols (Lang and Ghassemian, 2003;
Mcgarvey and Croteau, 1995). The plant produced isoprenoids -carotene (provitamine A)
and -tocopherol (VitE) are required for the maintenance of human health (Shintani and
Dellapenna, 1998; Hirschberg, 1999). Industrial uses of isoprenoids include products such as
colorants, fragrance, and flavorings (Lang and Croteau, 1999).
Otto Wallach who in 1910 recognized that isoprene is the basic constituent of terpens (Fig. 1)
and Leopold Ruzicha found that isoprene is the basic element for the synthesis of many
natural compounds including steroids. He postulated the biogenic isoprene rule, according
to which all terpenoids (derivatives of terpens) are synthesized via a hypothetical precursor
which he named active isoprene. This speculation was verified by Feodor Lynen in 1964,
when he identified isopentenyl pyrophosphate to be the active isoprene (Heldt, 2005).
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Terpenoids and Gibberellic Acids Interaction in Plants 349
3. Biosynthesis of gibberellins
Farmers in Asia were aware of a disease of rice plants called bakanae (foolish seedling)
disease. Infected plants would grow excessively taller than normal healthy plants, and being
fall over and be unharvestable. This disease was found to be caused by a fungus known as
Gibberella fujikuroi (Yabuta, T., 1935). Thus, Work on this special disease in Japan occurred by
Japanese scientists who isolated a growth promoting substances from cultures of a fungus
that parasitizes rice plants in the 1930s. They called it gibberellin. Scientists in the 1950s
rediscovered this work and extracted a range of chemical that elicit the growth response in
rice seedlings. Since that time, more than 100 slightly different gibberellins have been
identified chemically.
Gibberellins (GAs) are diterpene plant hormones or plant growth regulator of vascular
plants, fungi, and bacteria that are biosynthesized through complex pathways and control
diverse aspects of growth and development, including seed germination, shoot elongation,
leaf expansion, pollen-tube growth, trichome, flower and fruit development, cell division,
cell elongation and floral transition (Olszewski et al., 2002; Razem et al., 2006). Among more
than a hundered GAs identified from plants (http://www.plant-hormones.info/
gibberelline-nomenclature.htm) , only a small number of them, such as GA1, GA3, GA4, and
GA7 are thought to function as bioactive (biologically active gibberellins) hormones.
Therefore, many non bioactive gibberellins exist in plants as precursors for the bioactive
forms or deactivated metabolism (Yamaguchi, 2008). The major bioactive GAs, commonly
have a hydroxyl group on C-3 , a carboxyl group on C-6 and a lactone between C-4 and C-
10 (Fig. 4). GAs has been identified frequently in a variety of plant species, implying that it
acts as a widespread bioactive hormone.
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350 Advances in Selected Plant Physiology Aspects
According to the nature of the enzymes involved, gibberellins biosynthesis pathways are
usually divided into three parts. Each of the three parts consists of several steps:
I- Biosynthesis of ent-Kaurene
Recentely, a second, MVA- independent pathway to isopentenyl pyrophosphate (IPP) via
glyceraldehydes 3-phosphate and pyruvate was discovered in a green alga (Schwender et
al., 1996). Isopentenyl pyrophosphate is further converted to gerany geranyl pyrophosphate
(GGPP) by two enzymes, IPP-isomerase and GGPP-synthase in plastids of higher plants
(Dogbo & Camara, 1987).
Gerany gerany pyrophosphate is cyclized to ent-copalyl pyrophosphate (CPP) and finally
to ent-kaurene. In higher plants these reactions are catalyzed by two enzymes, CPP
synthase formerly Kaurene synthase A and ent-Kaurene (K) synthase formerly Kaurene
synthase B, renaming was suggested by Mcmillan 1979. Both enzymes were localized in
isolated proplastids of meristematic shoot tissues, but not in mature chloroplasts of pea
and wheat (Aachet al., 1995, 1997) or of pumpkins endosperm (Simcox et al., 1975; Aach et
al., 1995).
II- From ent-Kaurene to GA12
In the second stage of GA biosynthesis (Fig. 5), the C-19 methy (CH3) group of Kaurene is
oxidized in three steps to give ent-kaurenoic acid (KA). These oxidations are catalyzed by
ent-kaurene oxidase (KO), which is multifunctional because it can catalyze all three
reactions. In Arabidopsis, KO is encoded by GA3, and mutation in this gene will again
produce severly dwarfed plant (Shinjiro, 2008).
The intermediate, second, part of the pathway is catalyzed by microsomal NADPH-
dependent cytochrome P-450 monooxygenases at the endoplasmic reticulum (Lange, 1998).
Thus, the precursor ent-kaurene must be translocated from the proplastids to the
endoplasmic reticulum, although nothing is known about the transport mechanism (Lange,
1998). Ent-kaurene is oxidized into GA12, via ent-kaurenol, ent kaurenal, entkaurenoic acid,
ent-7 -hydroxy kaurenoic acid, and GA12-aldehyde. Certain biosynthesis steps, Such as 7-
oxidation, 12 -hydroxylation, and 13-hydroxylation are catalyzed by both monooxygenases
and soluble dioxygenases, which occasionally occur together within the same species or
even within the same tissue (Lange & Graebe, 1993).
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Terpenoids and Gibberellic Acids Interaction in Plants 351
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352 Advances in Selected Plant Physiology Aspects
cannabis plants. However, the regulation of the biosynthesis of terpenoids in plant cell is
poorly understood. We do not know how the metabolite fluxes between the primary and
secondary metabolisms are orchestrated. Plant growth regulators have essential roles in
growth and development of plants and plant resposes to environment. In order to understand
how interact with terpenoid biosynthesis; we focused on the role of GA3 in the regulation of
primary and secondary terpenoid production in Cannabis sativum at vegetative stage.
Treatment with exogenous GA3 resulted in a decrease of DXS activity in comparison with
the control plants (Fig. 6). Although, there is no report on the effect of GA3 on DXS activity,
many reports support a regulatory role of DXS for the production of MEP-derived
isoprenoids in plants (Rodriguez-concepcion, 2006). Estevez et al (2001) reported that
transgene mediated up regulation or down regulation of DXS levels in Arabidopsis were
correlated with concomitant changes in the levels of MEP derived isoprenoid end product.
The effects of the concentration of GA3 on the chlorophyll and carotenoid contents of
cannabis leaf are present in Fig. 7 & 8. Reduction in chlorophyll content after GA3
application has been reported in wheat (Misra & biswal, 1980), rice seedlings (Yim et al.,
1997) and pea (Bora & Sarma, 2006) which are consistence with our results (Mansouri et al.,
2011).The variation in the level of the cartenoids were similar to those observed for the
chlorophylls. Apparently carotenoids and chlorophylls accumulation are controlled through
a similar mechanism, because both of them are reduced by GA3. The changes in chlorophyll
and carotenoid were parallel with changes in DXS activity. It can show the limiting role of
DXS activity in chlorophyll and carotenoid synthesis.
The tocopherol is lipophilic antioxidants that are synthesized by photosynthetic
organisms, occurring mainly in leaves and seeds. Literature sources indicate that the
major tocopherol form in leaf tissues is tocopherol (Szymanska and Kruk, 2008).
Abdul Jaleel et al. (2007) reported that GA3 treatment stimulated tocopherol
accumulation in Catharanthus roseus.The tocopherolcontent of cannabis plants
decrease in 50 M GA3 treatment and increased with increasing GA3 at vegetative stage
(Fig. 9) which is consistence with other reports.
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Terpenoids and Gibberellic Acids Interaction in Plants 353
Fig. 7. Effects of gibberellic acid (GA3) on chlorophylls a, b and total in the leaves of
cannabis plants.
Fig. 8. Effects of gibberellic acid (GA3) on carotenoids in the leaves of cannabis plants.
Fig. 9. Effects of gibberellic acid (GA3) on a-tocopherol content in the leaves of cannabis plants
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354 Advances in Selected Plant Physiology Aspects
Exogenous GA3 caused an increasing in HMGR activity (Fig. 10). Since HMGR is an
important control point for the MVA pathway in plants (Kato-Emori et al., 2001), the
increase in HMGR activity should result in increasing the supply of phytosterols. Squalene
and phytosterol contents showed similar changes and increased concomitant with HMGR
activity in cannabis plants (Fig.11, 12). Consistent with our results, pea seedlings treated
with GA3 showed an increase in the HMGR activity (Russell & Davidson, 1982).
The amount of THC and CBD increase with increasing GA3 treatment in comparison with the
control plants (Fig. 13).The THC content was higher than those of CBD content ( Mansouri et al.,
2011). Perhaps, the increase observed in the THC and CBD content at high level of GA3 is not a
direct effect of GA3 treatment and could reflect the GA3 interaction with other plant hormones.
As has been shown that exogenous application of GA3 caused a clear increase in ACC content.
ACC oxidase activity and ethylene biosynthesis occur during the breaking of dormancy and
onset of germination in Fagus sylvatica L. seeds (Calvo et al., 2004). Furthermore, it is possible
that ethylene caused the increase observed in THC and CBD content.
Fig. 11. Effects of gibberellic acid (GA3) on squalene content in the leaves of cannabis plants.
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Terpenoids and Gibberellic Acids Interaction in Plants 355
Fig. 12. Effects of gibberellic acid (GA3) on phytosterol content in the leaves of cannabis
plants.
On the whole, these results showed that GA3 treatment had opposite effect on primary
terpenoid biosynthesis by MVA and MEP pathways. GA3 treatment caused a decrease in
DXS activity and biosynthesized primary terpenoids from MEP pathway, but this treatment
increased HMGR activity and phytosterols from MVA pathway. Whereas, secondary
terpenoids showed different response to GA3 treatment and it could be because of
interference of two biosynthetic pathways in their formation.
Fig. 13. Effects of gibberellic acid (GA3) on THC and CBD content in the leaves of cannabis
plants.
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356 Advances in Selected Plant Physiology Aspects
pathways, we studied the response of the main end products of these pathways and
cannabinoids under GA3 treatment in cannabis plants.
Effects of different concentrations of GA3 on levels of chlorophyll, carotenoids and
tocopherol in leaves of male and female cannabis plants were investigated. Male plants
treated with 50uM GA3 had lower chlorophyll a and total contents compared to the control
(Fig. 14A). Treatment of female leaves with 50 and 100uM GA3 significantly decreased
chlorophyll a, b and total contents in a dose-dependent pattern (Fig. 14B). The carotenoid
contents of treated plants were lower compared with the control (Fig. 15A). Low
concentrations of GA3 were more effective on decreasing carotenoid contents in male plants.
The phytyl (C20) conjugates chlorophylls and tocopheroles, and carotenoids (C40) are
produced by the MEP pathway. GA3 treatment decreased chlorophyll contents in C.sativa
plants. Reduction in chlorophyll content after GA3 application has been reported in, wheat
(Misra & Biswal 1980), peach trees (Monge et al. 1994), rice seedlings (Yim et al. 1997) and
pea (Bora & Sarma 2006). Perez et al. (1974) have shown that a mutant of tomato with
increased levels of GA3 contains less chlorophyll. Our results showed that GA3 caused a
decrease in carotenoid contents (Fig. 14). Apparently carotenoid and chlorophyll
accumulationis controlled through a similar mechanism, because both of them are reduced
by GA3. The previous studies indicate that GA3 treatments delayed the chloroplast
chromoplast conversion of colored fruit (Goldshmidt, 1998; Pfander, 1992). The
development of chromoplasts is accompanied by the accumulation of carotenoids (Vainstein
et al., 1994). Also by Rodrigo and Zacarias (2007) reported that GA3 reduced the ethylene
induced expression of early carotenoid biosynthetic genes and the accumulation of
phytoene in orange.
Fig. 15B shows the effect of different concentrations of GA3 on tocopherol content all
concentrations of GA3 sprayed on leaves caused an increase in tocopherol content in
both sexes. The highest increase was detected with 50uM GA3 in male plants. Apparently,
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Terpenoids and Gibberellic Acids Interaction in Plants 357
the pattern of changes in tocopherol content was an invert of that in chlorophyll and
carotenoid contents.
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358 Advances in Selected Plant Physiology Aspects
regulation of DXS levels in Arabidopsis were correlated with concomitant changes in the
levels of MEP-derived isoprenoid end products. However, in our investigation, the decrease
in the entire MEP pathway end product. The results showed an increase in tocopherol
content and a decrease in chlorophyll and carotenids. These data support this hypothesis
that several enzymes share control over the metabolic flux through the MEP pathway. The
MEP pathway might be regulated at several control points to compensate for fluctuations in
precursor/product equilibrium and redistribute the balance of control within the pathway
(Enfissi et al., 2005). Another possibility for this uncoordinated activity between the DXS
and the plastidic isoprenoids is the exchange or precursors between the cytosol and the
plastid. Strong biochemical evidence for such exchange of precursors was reported by
Kasahara et al., (2002), Nagata et al. (2002) and Hemmerlin et al. (2003).
Gibberellic acid treatment increased HMGR activity in the treated plants (Fig. 16). However
some differences are obvious between the two groups of plants of different sexes (Fig.16). The
male individuals had a greater HMGR activity at lower GA3 concentrations than female plants.
Activity of 3-hydroxy-3-methyglutaryl coenzyme A reductase (HMGR) in cannabis plants
demonstrated that HMGR activity was greater in plants treated with GA3. Consistent with
our result, Russell and Davidson (1982) reported that GA3 increased HMGR activity in pea
seedlings. It was shown that higher levels of HMGR activity were usually associated with
rapidly growing parts of plants (Brooker and Russell 1975). On the othe words, Ga is widely
regarded as a growth-promoting compound. Thus, it can be a candidate for stimulating of
the HMGR activity. The lower HMGR activity was seen in the male plants treated with 100
M GA3. The decrease observed in the HMGR activity could reflect the GA3 interaction in
high concentration with other plant hormones. As it has been shown that exogenous
application of GA3 caused a clear increase in ACC content, ACC oxidase activity and
ethylene biosynthesis occur during the breaking of dormancy and onset of germination in
Fagus sylvatica seeds (Calvo et al. 2004). In addition, it is possible that ethylene caused the
decrease observed in HMGR activity.
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Terpenoids and Gibberellic Acids Interaction in Plants 359
The mature leaves of male and female cannabis plants were used to determine the effect of
GA3 on squalene (biosynthetic precursor to all steroids), campestrol, stigmasterol, and
sitosterol (the most representative phytosteroids of the MVA pathway). A significant
increase in squalene (Fig. 17), stigmasterol and sitosterol accumulation occurred in female
and male plants treated with gibberellic acid (Fig. 18). The changes in the amount of
squalene and sitosterol were coordinate with the changes in HMGR activity. In addition,
GA3 had a stimulatory effect on campestrol accumulation in male plants.
The data in our study showed that GA3 treatment increased squalene and phytostrol
contents in a pattern similar to the changes in the HMGR acitivity. Since HMGR is an
important control point for the MVA pathway in plants (Kato-Emori et al. 2001), the increase
in HMGR activity should result in increasing the supply of phytostrols. Free sterols are
found predominantly in cell membranes and are thought to contribute to the proper
functioning of membranes by controlling the fluidity characteristics of the membrane
(Devarenne et al. 2002). Douglas and paleg (1974) demonstrated that the inhibitors of GAs
biosynthesis caused a decrease in sterol accumulation. Huttly and Philips (1995) suggested
that GA3 causes an increase in cell number and size to produce a significant effect. Since
these processes need the production of cell membrane. It can be assumed that GA3 should
induce the phytostrol biosynthesis to influence its effects on growth in plants.
A comparison between male and female plants showed that females had higher amounts of
THC; especially in the flowers (Fig. 19). THC content of the leaves was slightly lower than
that of flowers. The application of GA3 to cannabis plants resulted in a decrease in THC
content. Gibberellic acid treatment had a stronger effect in decreasing THC content in the
flowers of male plants in comparison with that of female plants. However, the leaves of the
two sexes indicated similar responses to GA3 treatment.
9-tetrahydrocannabinol (THC) is the cannabinoid responsible for the main psychoactive
effects of most Cannabis drug preparations (Mecoulam 1970). Factors that control
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360 Advances in Selected Plant Physiology Aspects
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Terpenoids and Gibberellic Acids Interaction in Plants 361
6. Conclusion
In conclusion, the pattern of changes in the amounts of primary terpenoids (chlorophyll,
carotenoids, and phytosterols) in Cannabis sativa suggest that GA3 have opposite effects on
the primary terpenoid biosynthesis of MEP and MVA pathways. In addition, the appearance
of the direct relationship between DXS and HMGR activity, and their main end products
confirm an important role for these enzymes in the MEP and MVA pathways regulation.
However, to understand the role of GA3 in terpenoid biosynthesis regulation, we need
further investigation.
7. Acknowledgement
The autor wishes to thank Mr. Hossein Mozafari phD student of Shahid Bahonar university
of Kerman for his critical comments and help of the manuscript.
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Advances in Selected Plant Physiology Aspects
Edited by Dr. Giuseppe Montanaro
ISBN 978-953-51-0557-2
Hard cover, 388 pages
Publisher InTech
Published online 25, April, 2012
Published in print edition April, 2012
The book provides general principles and new insights of some plant physiology aspects covering abiotic
stress, plant water relations, mineral nutrition and reproduction. Plant response to reduced water availability
and other abiotic stress (e.g. metals) have been analysed through changes in water absorption and transport
mechanisms, as well as by molecular and genetic approach. A relatively new aspects of fruit nutrition are
presented in order to provide the basis for the improvement of some fruit quality traits. The involvement of
hormones, nutritional and proteomic plant profiles together with some structure/function of sexual components
have also been addressed. Written by leading scientists from around the world it may serve as source of
methods, theories, ideas and tools for students, researchers and experts in that areas of plant physiology.
How to reference
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some-terpenoids-and-gibberellic-acid-in-cannabis-sativa-at-vegetative
Introduction
By natural products, we mean the molecules of nature. Of course, all life is made of molecules, and OH
we will not be discussing in great detail the major biological molecules, such as proteins and nucleic HO NHMe
acids, which we looked at in Chapters 49 and 50. In this chapter we shall talk much more about mol-
ecules such as adrenaline (epinephrine). Adrenaline is a human hormone. It is produced in
moments of stress and increases our blood pressure and heart rate ready for fight or flight. Youve HO
adrenaline
got to sit an exam tomorrowsurge of adrenaline. To an organic chemist adrenaline is intensely
interesting because of its remarkable biological activitybut it is also a molecule whose chemical
reactions can be studied, whose NMR spectrum can be analysed, which can be synthesized, and coniine
which can be imitated in the search for new medicines. an alkaloid
N
By the end of this chapter we hope you will be able to recognize some basic classes of natural H
products and know a bit about their chemistry. We will meet alkaloids such as coniine, the molecule
in hemlock that killed Socrates, and terpenes such as thujone, which was probably the toxin in
absinthe that killed the nineteenth-century artists in Paris. O
Then there are the ambiguous natural products such as the steroid cholesterol, which may cause thujone
a terpene
innumerable deaths through heart disease but which is a vital component of cell walls, and the
polyketide thromboxane, one drop of which would instantaneously clot all the blood in your body
but without which you would bleed to death if you cut yourself.
1414 51 . Natural products
H CO2H
O
H H O
Before moving on, just pause to
admire brevetoxin, a wonderful HO OH
and deadly molecule. Look at the cholesterola steroid thromboxane A2a polyketide
alternating oxygen atoms on the
top and bottom faces of alternate We will look at the structural variety within these four important classes and beyond, from
rings. Look at the rings perhaps the smallest natural product, nitric oxide, NO (which controls penile erections in
themselvessix-, seven-, and
eight-membered but each with
men), to something approaching the largestthe polyketide brevetoxin, the algal product in
one and no more than one oxygen red tides, which appear in coastal waters from time to time and kill fish and those who eat the
atom. Trace the continuous fish.
carbon chain running from the
O
lactone carbonyl group in the HO
bottom left-hand corner to the
Me H
aldehyde carbonyl in the top right.
There is no break in this chain brevetoxina toxic polyketide O O H
H
and, other than the methyl groups,
no branch. With 22 stereogenic H
H
centres, this is a beautiful piece of Me Me H
Me O O H
molecular architecture. If you want H Me H O
H
to read more about brevetoxin, O O
read the last chapter in Nicolaou H
and Sorensens Classics in total O O
synthesis, VCH, 1996. H H H Me
O O O
H H H
H H H Many natural products are the source of important life-saving drugsconsider the millions of
R N S lives saved by penicillin, a family of amino acid metabolites.
O N
O
CO2H
Natural products come from secondary metabolism
penicillin The chemical reactions common to all living things involve the primary metabolism of the big four
e.g. penicillin G; R = PhCH2
we met in Chapter 49nucleic acids, proteins, carbohydrates, and lipids. Now we must look at
chemical reactions that are more restricted. They occur perhaps in just one species, though more
commonly in several. They are obviously, then, not essential for life, though they usually help sur-
vival. These are the products of secondary metabolism.
The exploration of the compounds produced by the secondary metabolism of plants, microor-
ganisms, fungi, insects, mammals, and every other type of living thing has hardly begun. Even so, the
variety and richness of the structures are overwhelming. Without some kind of classification the task
of description would be hopeless. We are going to use a biosynthetic classification, grouping sub-
stances not by species but by methods of biological synthesis. Though every species is different, the
basic chemical reactions are shared by all. The chart on p. 1415 relates closely to the chart of primary
metabolism in the previous chapter.
secondary
metabolism
CO2
HO
O
photo- in O P H
synthesis plants
O
HO O
HO
O OH
HO alkaloids
HO erythrose-3-phosphate nitrogen compounds
OH OH
glucose
CO2H
O OH
P
aromatic compounds
O O amino acids, pigments, etc.
HO OH
OH
OH
O shikimic acid
phosphoenolpyruvate
O O O
polyketides
O R SCoA
n
OH linear polyketide chain
fatty acids
O
pyruvic acid O O
O SCoA
O
malonyl coenzyme A
SCoA
acetyl
coenzyme A OH terpenes
HO2C
OH
mevalonic acid steroids
citric
acid R CO2H
cycle
H NH2 alkaloids
aliphatic
amino acids nitrogen compounds
HO2C CO2H
HO CO2H
citric acid
chemical reaction in the usual sense: the starting material is incorporated into the product
1416 51 . Natural products
political adversaries to avoid any trouble in the future. Now, we would simply say that they are basic
because they are amines. Here is a selection with the basic amino groups marked in black.
HO
Me N
O
O
N O OH
H NMe
Me H H
N
HO
nicotine morphine atropine
Natural products are often named by a combination of the name of the organism from which they are
isolated and a chemical part name. These compounds are all amines so all their names end in -ine. They
appear very diverse in structure but all are made in nature from amino acids, and we will look at three types.
Solanaceae alkaloids
The Solanaceae family includes not only deadly Atropine is a racemic compound but the (S)-enantiomer
nightshade (Atropa belladonnahence atropine) plants occurs in henbane (Hyoscyamus niger ) and was given a
but also potatoes and tomatoes. Parts of these plants different name, hyoscyamine, before the structures were
also contain toxic alkaloids: for example, you should not known. In fact, hyoscyamine racemizes very easily just on
eat green potatoes because they contain the toxic heating in water or on treatment with weak base. This is
alkaloid solanine. probably what happens in the deadly nightshade plant.
H
H
N
H
H
H H the very toxic alkaloid
solanine, a mixture of
RO glycosides with R =
glucose, mannose, etc.
pyrrolidine
alkaloids
Further labelling experiments along these lines showed that the CO2H group as well as the Both reagents SAM and acetyl CoA
were discussed in Chapter 50. We will
amino group was lost from ornithine and that the rest of the molecule makes the pyrrolidine ring. not be able to repeat at length the
details of the chemistry of these and
The three-carbon side-chain in hygrine comes from acetate, or rather from acetyl CoA, and the N- other common biochemical reagents
methyl group comes from SAM. We can now work through the biosynthesis. already discussed there. In general, in
this chapter we will give only the
The first step is a pyridoxal-catalysed decarboxylation of ornithine, which follows the normal distinctive or interesting steps and
sequence up to a point. leave you to consult Chapter 50 if you
O need more help.
N N N
pyridoxal H
CO2H phosphate
H2N OH OH OH
PO PO PO
H NH2
N Me N Me N Me
H H H
Now the terminal amino group is methylated by SAM and the
secondary amine cyclizes on to the pyridoxal imine to give an ami-
Notice that the methylation step means that the two carbon atoms that
nal. Decomposition of the aminal the other way round expels eventually become joined to nitrogen in the five-membered ring remain
pyridoxamine and releases the salt of an electrophilic imine. different throughout the sequence. If, say, putrescine had been an
Me intermediate, they would not now be distinguishable.
N H
N NH H2N
H N H Enz
Me
SAM OH
OH
OH PO PO
PO N +
N Me Me N Me
N Me
H H
H
The rest of the biosynthesis does not need pyridoxal, but it does need two molecules of acetyl
CoA. In Chapter 50 we noted that this thiol ester is a good electrophile and also enolizes easily. We
need both reactivities now in a Claisen ester condensation of acetyl CoA.
Enz H B Enz OH
O O O OH O
Claisen ester
H condensation
O
CoAS CoAS CoAS CoAS
enolization
CoAS acetoacetyl CoA stable delocalized enol
The new keto-ester is very like the acetoacetates we used in Chapter 27 to make stable enolates
and the CoA thiol ester will exist mainly as its enol, stabilized by conjugation.
This enol reacts with the imine salt we have previously made and it will be easier to see this reaction
if we redraw the enol in a different O O
conformation. The imine salt does not
have to wait around for acetoacetyl N N
CoA to be made. The cell has a good Me Me
stock of acetyl CoA and its condensa- O SCoA O SCoA
tion product. H B Enz
1418 51 . Natural products
All that remains to form hygrine is the hydrolysis of the CoA thiol ester and decarboxylation of
the keto-acid. This is standard chemistry, but you should ensure that you can draw the mechanisms
for these steps.
O O O
N N N
thiol ester -keto-acid
Me hydrolysis Me decarboxylation
Me
O SCoA O OH hygrine
Tropinone is made from hygrine and it is clear what is needed. The methyl ketone must
enolize and it must attack another imine salt resembling the first but on the other side of the ring.
Such salts can be made chemically by oxidation with Hg(II) and biologically with an oxidizing
enzyme and, say, NAD+. The symbol [O] represents an undefined oxidizing agent, chemical or
biological.
O O OH Me N
[O] H
N N N O
H oxidation enolization cyclization
Me Me Me
tropinone
This complex route to tropinone was imitated as long ago as 1917 in one of the most celebrated
The cyclization step looks
dreadful when drawn on a flat reactions of all time, Robinsons tropinone synthesis. Robinson argued on purely chemical grounds
molecule, but it looks much that the sequence of imine salts and enols, which later (1970) turned out to be Natures route, could
better in the conformation of be produced under natural conditions (aqueous solution at pH 7) from a C4 dialdehyde, MeNH2
tropinone shown below.
and acetone dicarboxylic acid. It worked and the intermediates must be very similar to those in the
Me biosynthesis.
N CO2H
Me N
OH CHO
pH 7
+ Me NH2 + O
O
CHO water
CO2H tropinone
MeO
N N N
MeO
isoquinoline OMe
OMe
benzyl isoquinoline papaverine
Labelling shows that these alkaloids come from two molecules of tyrosine. One must lose CO2
and the other NH3. We can easily see how to divide the molecule in half, but the details will have to
wait a moment.
13C label
MeO
CO2H Papaver
somniferum N
MeO
H NH
2 OMe
HO
Tyr
tyrosine papaverine
OMe
The question of when the extra OH groups are added was also solved by labelling and it was
found that dihydroxyphenyl pyruvate was incorporated into both halves but the dihydroxyphenyl-
alanine (an important metabolite usually called dopa) was incorporated only into the isoquinoline
half.
HO CO2H Papaver
somniferum MeO
H NH
2 N
HO
dopa MeO
OMe
HO CO2H Papaver
somniferum
O OMe
HO
dihydroxyphenylpyruvate
The amino acid and the keto-acid are, of course, related by a pyridoxal-mediated transaminase
and the hydroxylation must occur right at the start. Both of these reactions are discussed in Chapter
50.
CO2H CO2H
pyridoxal
H NH2 transaminase O
HO HO
Tyr
tyrosine hydroxylase
H NH2 transaminase O
HO HO
dopa dihydroxyphenylpyruvate
1420 51 . Natural products
Catecholamines
Dopa and dopamine are important compounds because they are the precursors oxidase (Chapter 50) hydroxylates stereospecifically at the benzylic position to
to adrenaline in humans. Decarboxylation of dopa gives dopamine, which an give noradrenaline (norepinephrine).
H H OH OH
HO NH2 benzylic HO NH2 HO NHMe
hydroxylation SAM
HO HO HO
dopamine noradrenaline adrenaline
(norepinephrine) (epinephrine)
HO
The family of hormones that includes adrenaline and noradrenaline is control the breakdown of stored sugars to release glucose and they
often called the catecholamines (catechol is 1,2-dihydroxybenzene). have a direct effect on blood pressure, heart rate, and breathing. The
The hormones are produced in the adrenal gland around the kidneys relative proportion of noradrenaline and its N-methylated analogue,
and regulate several important aspects of metabolism: they help to adrenaline, controls these things. HO
catechol
Pyridoxal-mediated decarboxylation of dopa gives dopamine and this reacts with the keto-acid to
form an imine salt. This is an open-chain imine salt unlike the cyclic ones we saw in the pyrrolidine
alkaloids, but it will prove to have similar reactivity.
HO
HO
dopamine
pyridoxal NH
NH2 HO
HO
HO2C
HO2C OH OH
CO2
DHPP O
OH OH
The imine salt is perfectly placed for an intramolecular electrophilic aromatic substitution
by the electron-rich dihydroxyphenyl ring. This closes the isoquinoline ring in a Mannich-
like process (Chapter 27) with the phenol replacing the enol in the pyrrolidine alkaloid bio-
synthesis.
HO HO
Even in biological electrophilic aromatic
substitutions, it is still important to H
remember to write in the hydrogen
NH NH
HO HO
atom at the place of substitution
(Chapter 22)! HO2C HO2C
OH OH
OH OH
HO HO
CO2
NH NH
HO HO
HO2C pyridoxal
OH OH
OH OH
The cyclization product is still an amino acid and it can be decarboxylated by pyridoxal. Now we
have something quite like papaverine but it lacks the methyl groups and the aromatic heterocyclic
ring. Methylation needs SAM and is done in two stages for a reason we will discover soon. The final
oxidation should again remind you of the closing stages of the tropinone route.
Alkaloids are basic compounds from amino acid metabolism 1421
MeO MeO
NH N
4 SAM MeO [O] MeO
OMe OMe
papaverine
OMe OMe
The reaction to make the isoquinoline ring can be carried out chemically under very mild condi-
tions providing that we use an aldehyde as the carbonyl component. Then it works very well with
The reaction also works with an
rather similar compounds. aryl pyruvic acid, but the
HO HO decarboxylation is more difficult
to organize without pyridoxal.
NH2 pH 6 NH
HO HO
CHO
25C
water O
O
O
O
The mechanism is straightforwardthe imine is formed and will be protonated at pH 6, ready for
the CC bond formation, which is both a Mannich reaction and an electrophilic aromatic substitution.
HO HO
Notice that it was not necessary
to protect the OH groupsthe
NH NH acetal on the lower ring is not for
HO HO
H protection, and this group
O O (methylenedioxy or dioxolan) is
present in many benzyl
isoquinoline alkaloids. It is
O O formed in nature by oxidation of
an MeO group ortho to an OH
Complex benzyl isoquinoline alkaloids are formed by radical coupling group on a benzene ring.
A more interesting series of alkaloids arises when benzyl isoquinoline alkaloids cyclize by radical
reactions. Phenols easily form radicals when treated with oxidizing agents such as Fe(III), and benzyl
isoquinoline alkaloids with free phenolic hydroxyl groups undergo radical reactions in an intra-
molecular fashion through a similar mechanism. Here are the details of some methylations of a class See Chapter 39.
of alkaloids closely related to papaverine.
HO MeO
NH NH
HO HO The names of the alkaloids
H 2 SAM H should not, of course, be learned,
OH OH but they are a convenient handle
for quick reference. The prefix
nor means without a methyl
norlaudanosoline OH norreticuline OMe group, in this case the N-Me
group, as you can see with
2 SAM norreticuline and reticuline.
1 SAM
MeO MeO
NH NMe
MeO HO
H H
OMe OH
Methylating only one phenol on each ring of norreticuline leaves the other one free for radical
coupling. Reticuline is oxidized in the plant to isoboldine by a radical cyclization with the formation
of a new CC bond.
MeO MeO
orthopara coupling
NMe NMe
HO [O] HO
H H
OH
OH
The new CC bond is marked in black and the free phenolic OHs in green. Notice the relationship
between them. The new bond is between a carbon atom ortho to one OH group and a carbon atom
para to the other. We shall see in all these phenolic couplings that the ortho and para positions are the
only activated ones (ortho/ortho, ortho/para, and para/para couplings are all possible). Oxidation
occurs at the phenolic hydroxyl groups, and the resulting oxygen radicals couple.
MeO
MeO
NMe
O NMe
H Enz H O
H H
H
isoboldine
ortho/
para
MeO radical
coupling MeO
O
O H Enz
Phenol coupling occurs chemically under oxidation with Fe(III). The most famous example is the
coupling of 2-naphthol to give binaphtholan ortho/ortho coupling. The stereochemistry of
binaphthyls like this was discussed in Chapter 45.
OH
Fe(III) O etc. OH
O OH
Similar phenol couplings have been attempted in the laboratory with compounds in the benzyl
isoquinoline series but the nitrogen atom interferes if it is at all basic. When it has a carbonyl sub-
stituent the reactions do work reasonably well, but the yields are poor. Nature is still much better at
this reaction than we are.
MeO nonbasic N MeO
N OEt N OEt
HO Fe(III) HO
O O
[K3Fe(CN)6]
MeO MeO
OH OH
Reticuline is also the source of the morphine alkaloids by ortho/para radical coupling. The roles of
the two rings are reversed this time and it is quite difficult to see at first how the structures are related.
Alkaloids are basic compounds from amino acid metabolism 1423
MeO
MeO
ortho/para coupling
NMe [O]
HO O
H
OH NMe
MeO intermediate
reticuline OMe for morphine
O alkaloids
A great deal has happened in this reaction, but the new CC bond (black) is ortho to the green oxy-
gen atom in the top ring and para to the green oxygen atom in the bottom ring, so ortho/para coupling
has occurred. To draw the reaction mechanism we need to draw reticuline in the right conformation.
MeO
OH
rotate over to this
side of molecule OMe
NMe O
[O]
NMe
H
MeO MeO
OH reticuline O
MeO MeO
Enz H O HO
H
oxidative
NMe aromatize
NMe
coupling
MeO MeO
O O
One of the two rings can re-aromatize but the other has a quaternary carbon atom so no proton
can be lost from this site. Instead, the OH group in the top ring adds in conjugate fashion to the
enone in the bottom ring.
MeO MeO MeO
HO
O O
O H Enz OH O
This intermediate gives rise to the important alkaloids codeine and morphine, which differ only
by a methyl group. Nature can remove methyl groups as well as add them.
MeO MeO HO
O O O
NMe NMe NMe
H H
MeO HO HO
O codeine morphine
1424 51 . Natural products
NH NMe NMe
HO MeO MeO
H H [O] H
HO MeO MeO
OH OH OH
boldine
norlaudanosoline
The coupling is correctly para in the bottom ring but is meta in the top ring. But there is no mis-
take (neither by the authors nor by Nature!)this structure is correct and it has been made by
para/para coupling.
O O O
para/para
coupling
MeO MeO MeO
O O H Enz OH
One of the rings has aromatized, but the other cannotthis should remind you of the morphine
biosynthesis. However, there is no nucleophilic OH group here capable of conjugate addition to the
enone so a rearrangement occurs instead. The new bond to the lower ring migrates across the top ring.
You might even say that the lower ring does an intramolecular conjugate addition on the upper ring.
O HO
HO
Enz H
NMe NMe
NMe MeO
MeO MeO
H H
H H
MeO
MeO MeO
OH boldine
OH OH
Fatty acids and other polyketides are made from acetyl CoA 1425
After the rearrangement there is a proton available to be lost and the cation can aromatize. The para
relationship in the original coupling product has become a meta relationship by rearrangement. You
should be able to recognize this rearrangement from Chapter 37: it is a dienonephenol rearrangement.
In rearrangements like these with cationic intermediates, the group that can best support a posi-
tive charge usually prefers to migrate. The reasons for this are discussed in Chapter 37. Here is a
purely chemical example of the same reaction, giving 82% yield in acidic solution. The bond that
migrates is marked in black.
MeO MeO
NMe NMe
HO HO
H2SO4
MeO MeO
HO
O
OMe MeO multifloramine
Fatty acids and other polyketides are made from acetyl CoA
The sections that remain in this chapter show how Nature can take a very simple moleculeacetyl
CoAand build it up into an amazing variety of structures. There are two main pathways from
acetyl CoA and each gives rise to two important series of natural products.
SCoA
O O HO
HO2C OH
HO SCoA
malonyl CoA mevalonic acid
We shall discuss these four types of compounds in the order shown so that we start with the sim-
plest, the fatty acids. You met these compounds in Chapter 49 as their glyceryl esters, but you now
need to learn about the acids in more detail and outline their biosynthesis. Compare the structures of
the typical fatty acids in the chart overleaf.
These are just a few of the fatty acids that exist, but all are present in our diet and youll find many
referred to on the labels of processed foods. You should notice a number of features.
They have straight chains with no branching
They have even numbers of carbon atoms
They may be saturated with no double bonds in the chain, or
They may have one or more C=C double bonds in the chain, in which case they are usually cis (Z)
alkenes. If there is more than one C=C double bond, they are not conjugated (either with the
CO2H group or with each other)there is normally one saturated carbon atom between them.
1426 51 . Natural products
12
saturated fatty acids 1
CO2H lauric acid
16 1
CO2H palmitic acid
18
1
CO2H stearic acid
18 9 1
CO2H oleic acid
18 15 12 9 1
CO2H linolenic acid
20
14 11 8 5 1
CO2H arachidonic acid
Palmitic acid (C16 saturated) is the most common fatty acid in living things. Oleic acid (C18
mono-unsaturated) is the major fatty acid in olive oil. Arachidonic acid (C20 tetra-unsaturated) is a
rare fatty acid, which is the precursor of the very important prostaglandins, thromboxanes, and
leukotrienes, of which more later.
The prevalence of fatty acids with even numbers of carbon atoms suggests a two-carbon building
block, the most obvious being acetate. If labelled acetate is fed to plants, the fatty acids emerge with
labels on alternate carbons like this.
OH biosynthesis OH
O
O
The green blob might represent deuterium (as a CD3 group) and the black blob 13C. In fact, the
reactions are more complex than this suggests as CO2 is also needed as well as CoA and it turns out
that only the first two-carbon unit is put in as acetyl CoA. The remainder are added as malonyl CoA.
If labelled malonyl CoA is fed, the starter unit, as it is called, is not labelled.
CoAS OH biosynthesis OH
O O
no label O
Malonyl CoA is made from acetyl CoA and CO2 carried, as usual, on a molecule of biotin
(Chapter 50). The first stage in the fatty acid biosynthesis proper is a condensation between
acetyl CoA (the starter unit) and malonyl CoA with the loss of CO2. This reaction could be drawn
like this.
O O CO2
SR
SR
SR
condensing O O
enzyme
O O
Fatty acids and other polyketides are made from acetyl CoA 1427
Notice that CO2 is lost as the new CC bond is formed. When chemists use malonates, we like to
make the stable enol using both carbonyl groups, condense, and only afterwards release CO2 (Chapter
26). Nature does this in making
acetoacetyl CoA during alkaloid SR NADPH SR
biosynthesis, but here she works differ-
ently. O O -ketoacyl-ACP OH O
reductase
The next step is reduction of the
ketone group.
This NADPH reaction is typically SR SR
stereo- and chemoselective, though the
3-hydroxyacyl-ACP
stereochemistry is rather wasted here as OH O dehydratase O
the next step is a dehydration, typical of
what is now an aldol product, and
occurring by an enzyme-catalysed E1cB SR NADPH SR
mechanism.
The elimination is known to be a cis enoyl-ACP
O reductase O
removal of H and OH and the double
bond is exclusively trans (E). Only later in the nonconjugated unsaturated fatty acids do we get Z-
alkenes. Finally, in this cycle, the double bond is reduced using another molecule of NADPH to give
the saturated side-chain.
Now the whole cycle can start again using this newly made C4 fatty acid as the starter unit and
building a C6 fatty acid and so on. Each time the cycle turns, two carbon atoms are added to the acyl
end of the growing chain.
All of the enzymes needed for one cycle are clumped together to form two large proteins
(ACP, the acyl carrier protein, and CE, the condensing enzyme) which associate in a stable dimer. You saw a smaller multienzyme
complex in Chapter 50 (p. 1395), but
The long side-chain passes the substrate from enzyme to enzyme so that synthesis can be continuous this one is much more complex. More
until the chain is finished and only then is the thiol ester hydrolysed. The chart on p. 1428 illustrates are being discovered all the time
Nature invented the production line well
this. before Henry Ford.
O O SH O S long flexible
pantothenic acid
side-chain on the
SCoA O acyl carrier protein
(ACP)
O SH S
cysteine residue
on the condensing
enzyme (CE) CE ACP ACP
CE
ketoacyl
reductase
SH S O SH S O
CE ACP CE ACP
Me Me
hydratase
enoyl
reductase
growing chain
O transferred to O O
cysteine residue
on CE
Me S O S
Me O
SH
S
HO SCoA
CE ACP
CE ACP
O O
multienzyme complex
is ready to start the
next cycle of acylation
Fatty acids and other polyketides are made from acetyl CoA 1429
O O
CO2
R SACP
R SCE SACP NADPH
+
condensing O O -ketoacyl-ACP
O O enzyme reductase
The second method makes Z-3,4-unsaturated acids by deconjugation from the E-2,3-unsaturated
acids catalysed by an isomerase while the acyl chain is still attached to ACP. This is an anaerobic
route as no oxidation is required (the double bond is already thereit just has to be moved) and is
used by prokaryotes such as bacteria.
R R H Enz R H H
work.
OH H Enz H B Enz
R
R R
SACP
O SACP SACP
O HO
H
H Enz
Enz B
You may think this a rather unlikely reaction, but the same thing can be done in the labora-
tory. If a simple unsaturated ester is converted into its lithium enolate and then reprotonated
with water, the major product is the ester of the Z-3,4-enoic acid. Yields and steroselectivities are
excellent.
R R H OH R H H
H H H R H R
OEt H OEt OEt
R
H
H O O OLi
i-Pr2N Li
1,2-strain
disfavoured 1,3-(allylic) strain
The third method is a concerted stereospecific removal of two adjacent hydrogen atoms from the
chain of a fatty acid after synthesis. This is an aerobic route as oxidation is required and is used by
mammals such as ourselves. The stereochemistry of the reaction is known from labelling studies to
be cis elimination.
H H O H O
H
H
H 9 1 SCoA 9 1 SCoA
R R
This oxidation involves a chain of reagents including molecular oxygen, Fe(III), FAD, and NAD+.
A hydroxylation followed by a dehydration or a sulfur-promoted dehydrogenation has been suggest-
ed for the removal of the hydrogen atoms. The chemical reaction corresponding to the biological
reaction has not yet been discovered.
18 9 1
CO2H oleic acid
18 12 9 1
CO2H linoleic acid
18 1
12 9 6 CO2H -linolenic acid
20 1
14 11 8 CO2H eicosa-8,11,14-
trienoic acid
20
14 11 8 5 1
CO2H arachidonic acid
Fatty acids and other polyketides are made from acetyl CoA 1431
The final product of this chain of eventsarachidonic acidis one of the eicosanoids,
so called because eicosa is Greek for twenty, and the systematic names for these com-
pounds contain eicosanoic acid in some form. The leukotrienes resemble arachidonic acid
most closely, the prostaglandins have a closed chain forming a five-membered ring, and the
thromboxanes resemble the prostaglandins but have a broken chain. All are C20 compounds
with the sites of the alkenes (C5, C8, C11, and C14) marked by functionality or some other structur-
al feature.
compounds synthesized from arachidonic acid
20
14 11 8 5 1
CO2H
HO
11 O 1
8 8 5 1
CO2H
5 CO2H
20 11 14 20
14
HO OH
leukotriene LTA4
prostaglandin F2
8 5 1
CO2H
O
11 thromboxane A2
O 14 20
OH
These compounds are all unstable and all are involved in transient events such as inflam-
mation, blood clotting, fertilization, and immune responses. They are produced locally and decay
quickly and are implicated in autoimmune diseases like asthma and arthritis. They are made by
oxidation of arachidonic acidyou can see this best if you redraw the molecule in a different con-
formation.
8 5 1 8 5 1
CO2H cyclo-
O CO2H
oxygenase
14 O 20
20 11 14
11
PGG2
O
arachidonic acid OH
The first step is a radical abstraction of a hydrogen atom from an allylic position by oxygen (per-
haps carried on an iron atom in a haem). The atom removed is between two alkenes so that the
resulting radical is doubly allylic.
O O
R R
H H H
20 20
11 14 11 14
arachidonic acid delocalized radical
This allylic radical captures a molecule of oxygen at C11 to form a new oxyradical. The reaction
occurs at one end of the delocalized radical so that the product is a conjugated diene and the new
alkene is trans (E).
1432 51 . Natural products
R R
H
O O
11 14 O 11 14
O
conjugated E,Z-diene
Now we need to resume the full structure of the intermediate because the oxyradical does an elaborate
addition to the C8 alkene and then to the newly formed diene to form a new stable allylic radical.
8 5 1
8 5 1
CO2H O CO2H
O 14 O
20 11 20
O 11 14
Three new stereogenic centres are created in this cyclization, at C8, C9, and C12, and all are under
full control both from the centre already present and from the way in which the molecule folds up
under the guidance of the enzyme. Now the allylic radical reacts with oxygen to give the unstable
hydroperoxide PGG2.
8 5 1 8 5 1
O CO2H O CO2H
O O 20
20 11 14
11 14
PGG2
O
O OH OH
This unstable prostaglandin has been isolated from sheep but, as it has a half-life of only 5 min-
utes, this is no trivial matter. Both weak OO bonds are now reduced enzymatically to give the first
reasonably stable compound, PGF2 (PG just means prostaglandin).
HO
8 5 1 8 5 1
O CO2H
CO2H
O 20
11 14 11 20
14
O HO
PGG2
OH OH
[H] prostaglandin F2
The best evidence for this pathway comes from labelled oxygen molecules. If a mixture of
16O16O (ordinary oxygen) and 18O18O is supplied to an organism making PGF2, the product
has either both black OHs as 16O or both as 18O but no molecules are formed with one 16O and one
18O. These isotopes are easily measured by mass spectrometry. Both black OHs then come from one
and the same molecule of oxygennot an obvious conclusion when you inspect the molecule of
PGF2, and thus good evidence for this pathway.
11 O 1
8 CO2H
oxygen removes a hydrogen atom at C7
5
to form a stable conjugated radical
lipoxygenase
20
8 5 1 14
leukotriene LTA4
CO2H
14
20 8 5 1
11 O CO2H
arachidonic acid cyclo-
oxygenase O
14 20
oxygen removes a hydrogen atom at C13 11
to form a stable conjugated radical O
PGG2
OH
The initially formed radical is stabilized by two double bonds in the same way as that we have just
seen and reacts with oxygen in the same way again to give a trans-alkene and a new hydroperoxide.
O OH O OH
8 1 8 1
5 CO2H 5 CO2H
lipoxygenase
14 14
20 20
11 11
arachidonic acid
The next step is something quite new. No new CC bond is formed: instead, the diene attacks the
hydroperoxide to give an epoxide and a fully conjugated triene. The new double bond is cis this time,
which is what we should expect from the conformation we have been using. This is LTA4 and all the
other leukotrienes are made from this compound.
H Enz
O OH
8 1
5 CO2H
Enz B H
leukotriene LTA4
H 14
20
11
The relatively recent discovery of these unstable molecules of incredibly powerful biological activ- Prostaglandins and leukotrienes have
appeared several times before in this
ity means that we by no means know all about them yet. They are very important to our well-being book, and you can read about aspects
and important medical advances are bound to follow from a better understanding. of their laboratory synthesis on pp.
686, 1229, 1268, and 883.
HO
HO OH
OMe O OH OR
orsellinic acid
Me OH daunomycin (R = sugar)
alternariol
You might immediately be struck by the extent of oxygenation in these compounds. The shikimic
acid route produced ArC3 compounds with at most one OH group in the para position and others
1434 51 . Natural products
added ortho to that first OH group. Here we have multiple oxygenation with a predominant 1,3 pat-
tern. If we try to arrange an acyl polymalonate product to make orsellinic acid, this is what we shall
need.
Me Me Me
enol to ketone carbonyl O
CO2H tautomerism CO2H condensation CO2H
HO OH O O O O
orsellinic acid
Merely by writing ketones instead of phenols and doing one disconnection corresponding to a
simple carbonyl condensation, we have reached a possible starting material which is a typical acyl
polymalonate product without any reductions. This is what polyketides are. The fatty acids are
assembled with full reduction at each stage. Polyketides are assembled from the same process but
without full reduction; indeed, as the name polyketide suggests, many are made without any reduc-
tion at all. This is the biosynthesis of orsellinic acid.
CO2
O a tetraketide = 4 acetate units
O
O2C
SCoA O O O
rotate
SCoA CO2H
3 malonyl CoA
acetate =
starter unit acyl polymalonate intermediate
Me Me Me
O
CO2H cyclization CO2H enolization
CO2H
O O O O HO OH
orsellinic acid
This route has been demonstrated by feeding 13C-labelled malonyl CoA to a microorganism. The
orsellinic acid produced has three 13C atoms only, seen by an M + 3 peak in the mass spectrum. The
location of the labels can be proved by NMR. The starter unit, acetate, is not labelled.
Me
O
orsellinic acid synthase CO2H
O2C
SCoA
Penicillium species
HO OH
= 13C
orsellinic acid
As the polyketide chain is built up, any of the reductions or eliminations from fatty acid biosyn-
thesis can occur at any stage. The simple metabolite 6-methyl salicylic acid (6-MSA) is made in the
microorganism Penicillium patulum, and it could come from the same intermediate as orsellinic acid
with one reduction.
Me Me
O Me
O
CO2H CO2H
reduction cyclization CO2H
O O HO O
linear tetraketide 6-methyl salicylic acid
Reduction to the alcohol or to the unsaturated acid or ketone would give the right oxidation level
and could occur as the chain is built, after it is completed, or after cyclization. In fact, reduction to
Aromatic polyketides come in great variety 1435
the conjugated unsaturated triketide occurs as the third acetate unit is added, just as the fatty acid
route would lead us to expect.
O O O O OH O O O
NADPH
SR SR SR
linear triketide
This intermediate cannot cyclize as it has a trans double bond and the ends cannot reach each
other. First, the double bond is moved out of conjugation with the COSR group, again as in the fatty
acids, except that here the new Z double bond moves into conjugation with the remaining keto
group.
H
O H O O
H
SR SR SR
H
O O O
E -alkene conjugated extended enol Z -alkene conjugated
with acid group with ketone group
Now the last chain extension occurs and the completed Z-tetraketide cyclizes to 6-methyl salicylic
acid. Chemically, we would prefer not to carry the unstable Z-enone through several steps, but
Nature controls these reactions very precisely.
O O O
chain redraw in new
extension conformation
SR SR
malonyl
O CoA O
Me O Me O Me O
O
aldol and
SR dehydration SR enolization SR
O O OH
This precise sequence was discovered only through very careful double labelling experiments and H
after the discovery of specific inhibitors for the enzyme. Since polyketides can be made from the acyl Me
HO
polymalonate pathway with or without reduction and elimination at any step, the number of possi- O
ble structures is vast. With more reduction, no aromatic ring can be formed: macrolide antibiotics H O
OH
such as brefeldin A come from this route. brefeldin A
If you examine this structure, you should be able to find a continuous carbon chain made from an
acetate starter unit and seven malonyl CoA units with full or partial reduction occurring after many
acylation steps.
HO HO
4-hydroxy cinnamic acid
from shikimic acid activated for polymalonate addition
1436 51 . Natural products
The most common sequence uses three malonyl CoA acylations followed by cyclization to a new
aromatic ring. The simplest type is exemplified by resveratrole, the compound in red wine that helps
to prevent heart disease. Each step in this sequence is a simple reaction that you have met before.
O O O O
starter unit
3 malonyl CoA redraw
SCoA
3 malonyl CoA
HO
O OH
aldol cyclization
dehydration
enolization
O decarboxylation
O OH
COSCoA
HO HO
resveratrole
A different cyclization leads to the flavones and anthocyanidins. Reaction of the stable enol from a
1,3-diketone with the thiol ester as electrophile results in acylation at carbon in the manner of the
Claisen ester condensation (Chapter 28) with loss of CoASH and the formation of a trihydroxyben-
zene ring.
H
O O O OH
starter unit cyclization
enolization
HO CoAS O HO HO OH
O
OH O H OH O a flavanone
Aromatization of the central oxygen heterocycle by oxidation leads to the flavones, which are yel-
low or orange depending on their substituents. Dehydration leads to the red or blue anthocyanidins,
pigments of flowers and fruit. This important group of molecules also includes plant growth hor-
mones and defence compounds.
flavones OH anthocyanidins OH
RO O HO O
OH
OH O OH
R = H; naringenin, R = glucose; naringin pelargonidin, pigment of raspberries,
a bitter substance from grapefruit peel geraniums, and red grape skins
Terpenes are volatile constituents of plant resins and essential oils 1437
The first step is the Claisen ester condensation of two molecules of acetyl CoA, one acting as an
enol and the other as an electrophilic acylating agent to give acetoacetyl CoA. We saw the same reac-
tion in the biosynthesis of the pyrrolidine alkaloids earlier in this chapter.
Enz H B Enz OH
O O O
Claisen ester
H condensation
O
CoAS CoAS CoAS
enolization
CoAS acetoacetyl CoA
The third molecule of acetyl CoA also functions as a nucleophilic enol and attacks the keto group
of acetoacetyl CoA. This is not a Claisen ester condensationit is an aldol reaction between the enol
of a thiol ester and an electrophilic ketone.
OH
O O O OH O
CoAS
SCoA aldol CoAS SCoA
acetoacetyl CoA
1438 51 . Natural products
We have drawn the product with stereochemistry even though it is not chiral. This is because one
of the two enantiotopic thiol esters is hydrolysed while this intermediate is still bound to the enzyme,
so a single enantiomer of the half-acid/half-thiol ester results.
O OH O H2O O OH O
enantioselective
CoAS SCoA hydrolysis HO SCoA
enantiotopic thiol esters HMG-CoA
3(S)-3-hydroxy-3-methylglutaryl CoA
The remaining thiol ester is more electrophilic than the acid and can be reduced by the nucle-
ophilic hydride from NADPH. Just as in LiBH4 reductions of esters (Chapter 24), the reaction does
not stop at the aldehyde level, and two molecules of NADPH are used to make the alcohol. This is
mevalonic acid. OH
O OH O O OH O O OH
NADPH NADPH
OH O O
O acylation
O
SCoA SR
SCoA
SCoA
O
O OH O O O O
CoAS SCoA SR
mevalonic acid presursor linear triketide
Mevalonic acid is indeed the true precursor of the terpenes but it is a C6 compound and so it must lose
a carbon atom to give the C5 precursor. The spare carbon atom becomes CO2 by an elimination reaction.
First, the primary alcohol is pyrophosphorylated with ATP (Chapter 49); then the CO2H group and the
tertiary alcohol are lost in a concerted elimination. We know it is concerted because labelling the
diastereotopic hydrogen atoms on the CH2CO2H group reveals that the elimination is stereospecific.
O OH O OH
ATP elimination
HB
PP indicates the pyrophosphate HO OH HO OPP OPP
group transferred from ATP. mevalonic acid HA
H B HA
isopentenyl pyrophosphate
Terpenes are volatile constituents of plant resins and essential oils 1439
So is isopentenyl pyrophosphate the C5 intermediate at last? Well, yes and no. There are actually
two closely related C5 intermediates, each of which has a specific and appropriate role in terpene
biosynthesis. Isopentenyl pyrophosphate is in equilibrium with dimethylallyl pyrophosphate by a OPP
isopentenyl pyrophosphate
simple allylic proton transfer.
This is again a concerted reaction and again we know that by proton labelling. One of the two
enantiotopic protons (HS in the diagram) is lost from the bottom face of the allylic CH2 group while
the new proton is added to the top face of the alkene. This is an anti rearrangement overall.
OPP
H+ added to top
face of bond H dimethylallyl pyrophosphate
H
HB HB
OPP OPP
HA H
S HR H A
HR
The stereochemical details are interesting in establishing the mechanism but not important to
remember. What is important is that the origin of the two methyl groups in dimethylallyl pyrophos-
phate is quite distinct and can easily be traced if you always draw the intermediates in the way we
have drawn them. We will now switch to 13C labelling to make the point.
O OH O OH
ATP H
HO OH HO OPP OPP
OPP
mevalonic acid H H
isopentenyl pyrophosphate dimethylallyl pyrophosphate
The two C5 intermediates now react with each other. The dimethylallyl pyrophosphate is
the better electrophile because it is allylic, and allylic compounds are good at both SN1 and SN2
reactions (Chapter 17). Isopentenyl pyrophosphate is the better nucleophile because it can react
through an unhindered primary carbon atom to produce a tertiary cation. This is what we have in
mind.
OPP
OPP
OPP
better (allylic) better (unhindered)
electrophile nucleophile stable tertiary cation
Though this idea reveals the thinking behind the reaction, in fact it does not go quite like this. The
product is one particular positional and geometrical isomer of an alkene and the cation is not an
intermediate. Indeed, the reaction is also stereospecific (discovered again by proton labelling, but we
will not give the rather complex details) and this too suggests a concerted process.
OPP
Though terpenes are made from
OPP OPP C5 units, they are classified in
H geranyl pyrophosphate C10 units. The monoterpenes are
the C10 compounds, the
Geranyl pyrophosphate is the starting point for all the monoterpenes. It is still an allylic
sesquiterpenes (sesqui is Latin
pyrophosphate and repeating the alkylation with another molecule of isopentenyl pyrophosphate for one-and-a-half) are the C15
gives farnesyl pyrophosphate, the starting point for the sesquiterpenes, and so on. compounds, the diterpenes are
the C20 compounds, and so on.
OPP
C15 compounds
OPP OPP
(sesquiterpenes)
H farnesyl pyrophosphate
As soon as we start to make typical cyclic monoterpenes from geranyl pyrophosphate we run into
a snag. We cannot cyclize geranyl pyrophosphate because it has a trans double bond! We could
cyclize the cis compound (neryl pyrophosphate), and it used to be thought that this was formed from
the trans compound as an intermediate.
1440 51 . Natural products
OH
X
OPP
geraniol from geraniums
It is now known that Nature gets round this problem without making neryl pyrophosphate.
nerol from neroli oil
An allylic rearrangement occurs to move the pyrophosphate group to the tertiary centre. This is an
OH
unfavourable rearrangement thermodynamically and probably occurs via the allyl cation and cata-
lysed by Mg(II). There is no longer any geometry about the alkene. The molecule can
now rotate freely about a single bond and cyclization can occur. Even if only a small
OH amount of the rearranged allylic pyrophosphate is present, that can rearrange and more
farnesol, also present in neroli oil
can isomerize.
OPP
OPP OPP OPP
rotate cyclization
about bond possible
limonene
H
The product here is limonenea
More interesting compounds come from the cyclization of the first formed cation. The remaining
terpene of the peel of citrus fruits. One alkene can attack the cation to form what looks at first to be a very unstable compound but which is
enantiomer occurs in lemon peelthe
other in orange peel. See Chapter 45. actually a tertiary carbocation with the pinene skeleton.
H
=
H
-pinene
The camphor skeleton looks as though it might be formed by cyclization of the wrong end of the
alkene on to the cation. This would certainly give the right skeleton but the intermediate secondary
cation is rather unlikely.
?
=
camphor O
There is a better route. The more likely cation formed on the way to pinene could rearrange to the
camphor cation. This is a known chemical reaction and is a simple 1,2-shift of the kind discussed
in Chapter 37. However the new cation is formed, addition of water and oxidation would give
camphor.
1. hydration
2. oxidation
=
camphor O
Steroids are metabolites of terpene origin 1441
In the sesquiterpene series, similar cyclizations lead to an amazing variety of products. After the
initial unfavourable allylic rearrangement of the pyrophosphate group, farnesyl pyrophosphate can
give a six-membered ring cation known as the bisabolyl cation.
OPP redraw
farnesyl pyrophosphate
This cation does many things but it takes its name from the three fairly random proton losses that
lead to the -, -, and -bisabolenes.
loss of green
proton
H
loss of brown
H proton
-bisabolene
-bisabolene
Many other reactions give even larger and more complex terpenes with a variety of functionaliza-
tion but we will treat only one group in detail. These compounds are so important to us that they are
given a different name.
H H H H
O HO
cortisone ergosterol
All share the skeleton of four fused rings, three six-membered and one five-membered and con-
ventionally lettered AD. Beyond the ring stereochemistry and some common oxygenation patterns
they share little else. Some (such as the female sex hormones) have an aromatic A ring; some have
side-chains on the five-membered ring.
1442 51 . Natural products
At first glance, it is not at all clear that steroids are terpenoid in origin. The 5n numbers are absent
cholesterol is a C27 compound while the others variously have 20, 21, or 23 carbon atoms. Studies with
labelled mevalonic acid showed that cholesterol is terpenoid, and that it is formed from two molecules
of farnesyl pyrophosphate (2 C15 = C30 so three carbon atoms must be lost). Labelling of one or other
of the methyl groups (two experiments combined in one diagram!) showed that two of the green car-
bon atoms and one of the black carbon atoms were lost during the biosynthesis.
OH
HO2C
OH OPP OPP
mevalonic acid
2 farnesyl
OPP pyrophosphate
farnesyl pyrophosphate
H H
HO cholesterol
It is not obvious how the two farnesyl pyrophospate molecules could be combined to make the
steroid skeleton, and the chemistry involved is extraordinary and very interesting. The first clues came
from the discovery of the intermediates squalene and lanosterol. Squalene is obviously the farnesyl
pyrophosphate dimer we have been looking for while lanosterol looks like cholesterol but still has all 30
carbon atoms.
2 farnesyl
NADPH
pyrophosphate
squalene
squalene cyclization
H
H
HO lanosterol
The three carbon atoms that are lost from lanosterol (C30) in its conversion to cholesterol (C27)
are marked with brown arrows. Now at least we know which carbon atoms are lost. But many ques-
tions remain to be answered.
How does farnesyl pyrophosphate dimerize so that two electrophilic carbon atoms (CH2OPP)
join together?
Why does the formation of squalene require the reducing agent NADPH?
How does squalene cyclize to lanosterol so that the very odd labelling pattern can be achieved?
Where do the three lost carbon atoms go?
How is the sterochemistry controlled?
Before we tell you the answers, be warned: prepare for some surprises, and be ready to hold back out-
right disbelief!
Steroids are metabolites of terpene origin 1443
presqualene pyrophosphate H
OPP
Maybe its not so obvious that this is more rational! The first CC bond formation is quite straight-
forward. The alkene in the red molecule attacks the allylic pyrophosphate in the black molecule in a
simple SN2 reaction. The product is a stable carbocation. Only one CC bond remains to be formed to
close the three-membered ring and this occurs by the loss of a proton from the black molecule.
OPP
H H R
R R R
R We will abbreviate the long terpene
R side-chain to R from now on.
H
PPO OPP
OPP
This is a very remarkable reaction. Such reactions do not occur chemically: this biological one
occurs only because the molecule is held in the right shape by the enzyme and because the new ring is
three-membered. Three-membered rings are very easily formed but also very easily openedand
that is what happens to this ring. In the presence of NADPH, a series of rearrangements gives a series
of carbocations, the last of which is trapped by reduction.
The first step is the migration of one of the bonds (shown in green) of the three-membered ring to
displace the pyrophosphate leaving group, expand the ring to four-membered, and release some
strain. Now the cyclobutyl cation breaks down to give an open-chain allylic cation stabilized by one
of the alkenes. This is the cation that is reduced by NADPH.
H H NADP
R R
R R R
R R R
OPP squalene
If you follow this sequence backwards, you will see that the originally formed rational bond
(shown in green) is the one that migrated and is retained in squalene, while the second bond is
cleaved in the last step.
This may all seem far-fetched, but it happens in laboratory reactions too! Treatment of the sim-
plest cyclopropyl alcohol with HBr gives cyclobutyl bromide by a similar rearrangement.
HBr Br Br
H H
H H
OH OH2
shown has been suggested as an intermediate. Make sure that you can draw mechanisms for each
starting material to give the intermediate cation and from the cation to each product.
cyclopropyl
methanol OH X
a
b
cyclobutanol OH X
b
a
c c
but-3-en-1-ol OH X
X
Squalene to lanosterol
The next step is simplethe epoxidation of one of the terminal double bondsbut it leads to two of the
most remarkable reactions in all of biological chemistry. Squalene is not chiral, but enzymatic epoxidation
of one of the enantiotopic alkenes gives a single enantiomer of the epoxide with just one stereogenic centre.
squalene
O
squalene oxide
We will start now to draw squalene in a coiled up way as the next step is the polycyclization of the
epoxide. The basic reaction is best seen first in the flat, though we will draw the stereochemistry
immediately. The first alkene cyclizes on to the epoxide and then each remaining alkene cyclizes on
to the next to give a stable tertiary cation.
H
H
O HO
H
H
By analogy with what has gone before, you might now expect a tame hydration or reduction of
this cation. Nothing of the sort! A rearrangement occurs in which five consecutive 1,2-shifts are fol-
lowed by an elimination. Since this reaction organizes the backbone of the steroids, it is often called
the steroid backbone rearrangement.
H
H Me
H H
Me
H Me Me
HO HO
H H lanosterol
Steroids are metabolites of terpene origin 1445
Finally, we have reached lanosterol. Now we will go back over these two steps and discuss them a
bit more. Consider first the regiochemistry of the cyclization. The epoxide opens in the way we
would expect to give positive charge at the more substituted carbon atom and then all the alkenes
attack through their less substituted end (again as we would expect to give positive charge at the
more substituted carbon atom)all except one. The third alkene cyclizes the wrong waythis is
presumably a result of the way the molecule is folded.
We learn much more about the folding by examining the stereochemistry of the product cation.
First, all of the stereochemistry of each alkene is faithfully reproduced in the product: the cyclization
is stereospecific. This is emphasized in colour in the diagram. The green stereochemistry arises
because the green Me and H were trans in the first alkene of squalene, the black Me and H trans in the
second, and the brown trans in the third. But what about the relationship between the green methyl
and the black H? Or between the black and brown methyls? These were determined by the folding
and the key observation is that all the relationships are trans except that between the green Me and
the black H. Now we can draw a conformation for the cyclization.
H chair
chair
H
Me H
Me Me Me
O H
H Me
R
HO H Me H
H Me
boat
When the transition state for a ring closure forms a chair then a trans relationship results. This is
the case for the black Me and brown Me. When a boat is formed a cis relationship results. This is the
case for the green Me and black H. Squalene folds up in a chairboatchair conformation and that
leads to the observed stereochemistry.
Next, we need to look at the stereochemistry of the rearrangement step. If we draw the product
cation as nearly as possible in the conformation of folded squalene, we will see which substitutents
are axial and which equatorial. H
cis relationship between Me R
and H stops chain of migrations Me
H H
Me Me
H Me
all anti migrations
HO
Me Me
H R HO
Me
H H lanosterol
Each group that migrates (black) is axial and is anti-periplanar to the one before so that each
migrating group does an SN2 reaction on the migration terminus with inversion. The chain stops
because of the cis relationship between the green Me and H in ring B and an elimination of the green
H is all that can happen.
The remainder of the biosynthesis of cholesterol requires various redox reactions and is a bit of an
anticlimax: the details are summarized in the scheme below.
H
lanosterol R cholesterol H
reduce alkene R
introduce alkene H
H
H
Me
H H
HO H HO
Me Me CO2
1446 51 . Natural products
H
H
H H
HO
The first step is the formation of a symmetrical allyl cation, which then initiates the cyclization. The
next double bond is disubstituted so that it has no built-in regioselectivity but prefers to form a six-
membered rather than a five-membered ring B. The next double bond is trisubstituted and directs the
formation of a six-membered ring C. The alkyne, being linear, can reach only through its inner end and
so a five-membered ring D is formed. The resulting linear vinyl cation picks up a molecule of water to
give the ketone via its enol.
H2O
OH
H H
H2O
H H H H
H
Problems 1447
The five-membered ring A is there to ensure efficient initiation of the cyclization by the symmet-
rical allylic cation. It can easily be opened with ozone and the product cyclized to progesterone.
O
O
H
H 1. O3
H H
H H 2. KOH
O
progesterone
The conformation of the molecule in the moment of cyclization can be seen easily by working
backwards from the product. The green dashed lines show new bonds that are being formed. All the
six-membered rings in the transition state are chairs and all the ring junctions trans. This is an
impressive result as there is no enzyme to help the molecule fold up in this way.
O
H
H H
By studying the chemistry that Nature uses in living things we can learn new reactions as well as new
ways in which to carry out known reactions. Many of the reactions in this chapter would be laughed at
by worldly wise chemists if they appeared in a research proposal, but they have been evolved over mil-
lions of years to do precise jobs under mild conditions. Humans have been doing complex organic
chemistry for only about a hundred years so that learning from Nature is one of the most important
ways in which organic chemistry is advancing at the beginning of the twenty-first century.
Problems
1. Assign each of these natural products to a general class (such as
amino acid metabolite, terpene, polyketide) explaining what
makes you choose that class. Then assign them to a more specific
part of the general class (for example, tetraketide, sesquiterpene).
Primula acaulis
OH
HO OH
Penicillium
OH
shikimic acid CH3CO2H
1448 51 . Natural products
N N
H H
CO2H pelletierine
HO OMe
orientaline stephanine
NH2 O
HO HO
tyrosine
HO CO2H HO CO2H
NH2 O
HO HO
DOPA
Problems 1449
9. Tetrahydrocannabinol, the major psychoactive compound in 12. Borneol, camphene, and -pinene are made in nature from
marijuana, is derived in the Cannabis plant from olivetol and geranyl pyrophosphate. The biosynthesis of -pinene and the
geranyl pyrophosphate. Details of the pathway are unknown. related camphor is described in the chapter. In the laboratory
Make some suggestions and outline a labelling experiment to bornyl chloride and camphene can be made from -pinene by the
establish whether your suggestions are correct. reactions described below. Give mechanisms for these reactions
and say whether you consider them to be biomimetic.
olivetol
ArOH
OH
H Cl
-pinene bornyl chloride camphene
tetrahydro-
cannabinol
H 13. Suggest a biosynthetic route to the monoterpene chrys-
O anthemic acid that uses a reaction similar to the formation of
squalene in steroid biosynthesis.
10. Both humulene, mentioned in the chapter, and caryophyllene Pyrethrum plants
are made in nature from farnesyl pyrophosphate in different plants.
OPP
Suggest detailed pathways. How do the enzymes control which CO2H
product is formed? chrysanthemic acid
How could the same route also lead to the natural products
H yomogi alcohol and artemisia ketone?
O
HO
H
yomogi alcohol artemisia ketone
humulene caryophyllene
14. In the chapter we suggested that you could detect an acetate
starter unit and seven malonate additional units in the skeleton of
11. Abietic acid is formed in nature from mevalonate via the
brefeldin. Give the mechanism of the addition of the first malonyl
intermediates shown. Give some more details of the cyclization
CoA unit to acetate. Draw out the structure of the complete acyl
and rearrangement steps and compare this route with the bio-
polymalonate chain and state clearly what must happen to each
synthesis of the steroids.
section of it (reduction, elimination, etc.) to get brefeldin A.
OPP OPP H
HO Me
O
H O
OH
brefeldin A
H H OAc
HO2C abietic acid
+
D.E. Vance and J.E. Vance (Ed~,.) Biochemistl3' (~['Lipid.~, Lil~Olnoteins aml Memhl~mes (4th E~hl.)
~-~ 20(12 Elsevier Science B.V. All rights reserved
CHAPTER 4
1. Introduction
Plants produce the majority of the world's lipids, and most animals, including humans,
depend on these lipids as a major source of calories and essential fatty acids. Like other
eukaryotes, plants require lipids for membrane biogenesis, as signal molecules, and as a
form of stored carbon and energy. In addition, soft tissues and bark each have distinctive
protective lipids that help prevent desiccation and infection. To what extent does the
biochemistry of plant lipid metabolism resemble that in other organisms? This chapter
mentions a number of similarities, but emphasizes aspects unique to plants. Major
differences between lipid metabolism in plants and other organisms are summarized in
Table 1.
The presence of chloroplasts and related organelles in plants has a profound effect
on both gross lipid composition and the flow of lipid within the cell. Fatty acid
Table 1
Comparison of plant, mammalian, and bacterial lipid metabolism
synthesis occurs not in the cytosol as in animals and fungi, but in the chloroplast
and other plastids. Acyl groups must then be distributed to multiple compartments,
and the complex interactions between competing pathways are a major focus of plant
lipid biochemists. It is also significant that the lipid bilayers of chloroplasts are largely
composed of galactolipids rather than phospholipids. As a result, galactolipids are the
predominant acyl lipids in green tissues and probably on earth.
Plant lipids also have a substantial impact on the world economy and human nutrition.
More than three-quarters of the edible and industrial oils marketed annually are derived
from seed and fruit triacylglycerols. These figures are particularly impressive given that,
on a whole organism basis, plants store more carbon as carbohydrate than as lipid. Since
plants are not mobile, and since photosynthesis provides fixed carbon on a regular basis,
plant requirements for storage lipid as an efficient, light weight energy reserve are less
acute than those of animals.
Finally, hundreds of genes required for plant lipid biosynthesis, utilization and
turnover have now been cloned. In addition to providing valuable information on
enzyme structure and function, these genes are being exploited to design new, more
valuable plant oils. The coordination of lipid metabolic genes with each other and with
their potential regulators may also become better understood, as DNA microarray and
other genomic technologies mature.
2.1. Plastids
Although all eukaryotic cells have much in common, the ultrastructure of a plant
cell differs from that of the typical mammalian cell in three major ways. The plasma
membrane of plant cells is shielded by the cellulosic cell wall, preventing lysis in the
naturally hypotonic environment but making preparation of cell fractions more difficult.
The nucleus, cytosol and organelles are pressed against the cell wall by the tonoplast,
the membrane of the large, central vacuole that can occupy 80% or more of the cell's
volume. Finally, all living plant cells contain one or more types of plastid.
The plastids are a family of organelles containing the same genetic material, a
circular chromosome present in multiple copies. Young or undifferentiated cells contain
tiny proplastids that, depending on the tissue, may differentiate into photosynthetic
chloroplasts, carotenoid-rich chromoplasts, or any of several varieties of colorless
leucoplasts, including plastids specialized for starch storage [1]. These different types
of plastids, which may be interconverted in vivo, have varying amounts of internal
membrane but invariably are bounded by two membranes. The internal structure of
chloroplasts is dominated by the flattened green membrane sacks known as thylakoids.
The thylakoid membranes contain chlorophyll and are the site of the light reactions of
photosynthesis.
As noted above, chloroplasts and other plastids are enriched in galactolipids (Fig. 1).
They also contain a unique sulfolipid, sulfoquinovosyldiacylglycerol, whose head
group is a modified galactose. The phospholipid components of plastids are less
95
L.o o
Pea Chloroplasts %
Sulfoc]uinorosyldiacylglycerol Thylakoids 4
c H2SO~ Inner membrane 4
Outer membrane 4
0 0 t Daffodil chromoplasts 5
Cauliflower proplastids 6
Potato leucoplasts 5
o
Fig. I. Composition of plastid membranes. Figures given are percentages (as % of total lipid) of the pictured
lipid in the membranes specified. Data from Harwood, J.L. (1980) Plant acyl lipids: structure, distribution
and analysis. In: EK. Stumpf (Ed.) The Biochemistry of Plants, Vol. 4, Academic Press, pp. 2-56 and
Sparace, S.A., Kleppinger-Sparace, K.F. (1993) Metabolism in non-photosynthetic, non-oilseed tissues. In:
T.S. Moore Jr. (Ed.) Lipid Metabolism in Plants, Boca Raton, FL, CRC Press, pp. 569-589.
._=
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o 0
~ ~ -~ ~.~
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97
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98
are produced for export to the rest of the cell. An acyl-CoA synthetase identified
on the outer membrane of plastids is thought to facilitate release of acyl groups
into the cytosol. It should also be noted that, although net lipid traffic is from the
plastids, this organelle can likewise be on the receiving end. In addition to small
quantities of plastidial phospholipids whose head groups are not known to arise in
that compartment, there may be considerable flow of extraplastidially constructed
diacylglycerol backbones into the galactolipid synthesis pathway. The quantitative
significance of this backflow depends on the plant species, as will be discussed in
Section 5.3.
The endoplasmic reticulum has traditionally been viewed as the primary source of
phospholipids in plant cells. With the exception of cardiolipin, all of the common
phospholipids can be produced by microsomal fractions. The endoplasmic reticulum
also serves as the major site of fatty acid diversification. Although plastids do have the
ability to synthesize polyunsaturated fatty acids, they are formed on acyl lipid substrates
and are not typically exported. Thus, the endoplasmic reticulum desaturation pathways
are of particular importance for developing seeds that store large quantities of 18 : 2
and 18:3. Pathways for the production of unusual fatty acids found primarily in seed
oils have likewise been described in microsomes. Not surprisingly, the endoplasmic
reticulum also appears to be instrumental in the formation of the triacylglycerols
themselves and the lipid bodies in which they are stored (Section 7).
2.3. Mitochondria
Next to plastids and the endoplasmic reticulum, the plant mitochondrion is probably the
organelle investigated the most thoroughly with respect to lipid metabolism. Its ability to
synthesize phosphatidylglycerol and cardiolipin is well established. Although most fatty
acids for mitochondrial membranes are imported from the plastids or the ER, recently
mitochondria have been shown to synthesize low levels of fatty acids from malonate.
Octanoate is a major product of this pathway and serves as a precursor for the lipoic
acid cofactor needed by glycine decarboxylase and pyruvate dehydrogenase [2].
Fatty acid synthases may be classified into two groups. 'Type I' fatty acid synthases
are characterized by the large, multifunctional proteins typical of yeast and mammals
(Chapter 6), while 'Type II' synthases of most prokaryotes are dissociable into compo-
nents that catalyze individual reactions (Chapter 3). Plants, while certainly themselves
eukaryotic, appear to have inherited a Type II fatty acid synthase from the photosynthetic
prokaryotes from which plastids originated.
The ground-breaking studies of Overath and Stumpf in 1964 [4] established not only
that the constituents of the avocado fatty acid synthesis system could be dissociated
and reconstituted, but also that the heat stable fraction from E. coli we now know
as acyl carrier protein (ACP) could replace the corresponding fraction from avocado.
Plant ACPs share both extensive sequence homology and significant elements of three-
dimensional structure with their bacterial counterparts. In plants, this small, acidic
protein not only holds the growing acyl chain during fatty acid synthesis, but also is
required for synthesis of monounsaturated fatty acids and plastidial glycerolipids.
Fatty acid synthase is generally defined as including all polypeptides required for
the conversion of acetyl and malonyl-CoA to the corresponding ACP derivatives, the
acyl-ACP elongation cycle diagrammed in Chapter 3, and the cleavage of ACP from
completed fatty acids by enzymes termed thioesterases or acyl-ACP hydrolases [5]. All
components of fatty acid synthase occur in plastids, although they are encoded in the
nuclear genome and synthesized on cytosolic ribosomes. Most of the 8-10 enzymes of
the pathway are soluble when isolated from homogenates. Nevertheless, some evidence
suggests that at least ACP and some subunits of acetyl-CoA carboxylase may be
associated with the plastid membranes.
Despite the presence of acetyl-CoA : ACP acyltransferase activity in plant fatty acid
synthase preparations, acetyl-ACP does not appear to play a major role in plant fatty
acid synthesis (Jaworski, 1992). Instead, the first condensation takes place between
acetyl-CoA and malonyl-ACE This reaction is catalyzed by 13-ketoacyl-ACP synthase
III, one of three ketoacyl synthases in plant systems (Fig. 2). The acetoacetyl-ACP
product then undergoes the standard reduction-dehydration-reduction sequence to
produce 4 : 0-ACE the initial substrate of ketoacyl-ACP synthase I. KAS I is responsible
for the condensations in each elongation cycle up through that producing 16:0-ACE
The third ketoacyl synthase, KAS II, is dedicated to the final plastidial elongation, that
of 16 : 0-ACP to 18 : 0-ACE
100
The major components of the long-chain acyl-ACP pool in most plant tissues are 16:0-
ACE 18 : 0-ACP and 18 : l-ACE This finding highlights the importance of stearoyl-ACP
desaturase, the plastidial enzyme responsible for Ag-desaturation in plants. In contrast to
the desaturation system of Escherichia coli (Chapter 3), the plant enzyme introduces the
double bond directly to the AV-position. Unlike yeast and mammalian A%desaturases,
it is a soluble enzyme and is specific for acyl-ACPs rather than acyl-CoAs (McKeon,
1982). In recent years, work on stearoyl-ACP desaturase has progressed rapidly and
is now providing a more detailed understanding of the fundamental mechanisms of
oxygenic fatty acid desaturation. Genes for the enzyme have been cloned from a number
of species, and the structure of the castor bean AV-desaturase has been determined to
2.4 ,~ resolution. A combination of the crystal structure and spectroscopic methods
has revealed two identical monomers, each with an active site containing a diiron-oxo
cluster. Reduction of the iron by ferredoxin leads to its binding of molecular oxygen.
The resulting complex ultimately removes electrons at the AV-position, resulting in
double bond formation [6].
Although the most common unsaturated fatty acids in plants are derived from oleic
acid, a wide range of unusual fatty acids are found in the seed oils of different species. Di-
vergent plastid acyl-ACP desaturases have been shown to account for some of this diver-
sity. For example, Coriandrum sativum achieves seed oils rich in A t'- 18 : 1 (petroselinic
acid) by desaturation of 16:0 at the A4-position followed by elongation, while Thun-
bergia alata attains a similar oil by direct A%desaturation of 18:0. Several structure-
function relationships suggested by unusual desaturases have been tested in the acyl-ACP
desaturase system. Shortening the acyl binding pocket of the A 9-18:0 desaturase by al-
tering a single amino acid as in Doxantha unguis-cati shifts substrate specificity in favor
of 16 : 0-ACP (Cahoon, 1998). In addition, a set of five specific amino acids suggested by
the Thunbergia gene transforms the castor bean AV-desaturase to a A%desaturase, while
enzymes with certain subsets of the five amino acids can desaturate at either position [6].
Among prokaryotes, all acyl groups exiting the dissociable fatty acid synthase are
transferred directly from ACP to polar lipids. However, plants must also release
sufficient fatty acid from ACP to supply the extraplastidial compartments. Since the
101
typical chloroplast exports primarily 18:1 and 16:0, the same fatty acids that comprise
the greatest fraction of long-chain acyl-ACPs, it might be assumed that a relatively
non-specific thioesterase releases 16- and 18-carbon fatty acids from ACE However,
molecular and biochemical analyses of cloned plant thioesterases suggest that plants
possess individual thioesterases with specificity either for 18 : 1 or for one or more
saturated fatty acids [7]. The most prominent thioesterase in most plants has a strong
preference for 18: 1-ACE making 18 : 1 the fatty acid most available for extraplastidial
glycerolipid synthesis. In contrast, mangosteen, a plant with seed oil particularly high in
18 : 0, contains an 18 : 0-ACP thioesterase gene that has been used to engineer rapeseed
with high 18:0 content (Hawkins, 1998).
Plants that synthesize certain unusual fatty acids have additional or modified
thioesterases. For example, several plant species that produce storage oils contain-
ing large amounts of 8- to 14-carbon acyl chains contain thioesterases specific for
those chain lengths. By removing acyl groups from ACP prematurely, the medium-
chain thioesterases simultaneously prevent their further elongation and release them for
triacylglycerol synthesis outside the plastids. In addition, both the standard A9-18:1
thioesterase and a A6-18 : 1 thioesterase have been purified from the A6-18 : 1 accumu-
lating coriander plant. Thus plants, by regulating expression of different thioesterases,
can both fine tune and radically modify the exported fatty acid pool.
The malonyl-CoA that supplies all but two carbons per fatty acid is produced from acetyl-
CoA and carbon dioxide by acetyl-CoA carboxylase (ACC). In plants, malonyl-CoA for
fatty acid synthesis is apparently provided by a plastid ACC, while a cytosolic ACC
contributes malonyl units for fatty acid elongation as well as synthesis of flavonoids,
polyketides, and other metabolites. As with fatty acid synthase, ACC forms may be cate-
gorized either as 'eukaryotic' enzymes, which are dimers of a multifunctional polypeptide
(Chapter 6), or 'prokaryotic' enzymes, which are heteromers of biotin carboxyl carrier
protein, biotin carboxylase, and two subunits of carboxyltransferase (Chapter 3). In the
grass family, both plastids and cytosol house 'eukaryotic' enzymes. However, dicots and
monocots other than grasses appear to have both forms, with the 'eukaryotic' form lim-
ited primarily to the cytosol, and 'prokaryotic' enzymes dominating in the plastids [8].
Assembly of the 'prokaryotic' form requires participation of both the nuclear genome,
which encodes biotin carboxyl carrier protein, biotin carboxylase, and the c~-subunit of
carboxyltransferase, and the plastid genome, which has retained the gene for the car-
boxyltransferase 13-subunit, perhaps due to a requirement for RNA editing (Sasaki, 2000).
In other kingdoms, ACC is a major control point for fatty acid biosynthesis. Although
the mechanisms acting in plants are incompletely characterized, there is evidence that
102
plant ACCs are also tightly regulated [9]. For example, both redox regulation via
thioredoxin and phosphorylation of the carboxyltransferase have been implicated in
up-regulation of the 'prokaryotic' ACC by light. Conversely, feedback inhibition is
observed at the level of ACC when tobacco cell cultures are given exogenous fatty acids.
Due to its impact on the rate of fatty acid synthesis, ACC is considered a promising
target in oilseed improvement programs, and some increases in oil content have been
obtained by engineering a cytosolic ACC gene to be expressed in rapeseed plastids.
In the plastids, acyltransferases provide a direct route for acyl groups from ACP to enter
membrane lipids. Since this is the standard pathway in E. coli and cyanobacteria, both
the enzymes of phosphatidic acid synthesis in plastids and the glycerolipid backbones
they produce are termed 'prokaryotic.' In both chloroplasts and non-green plastids,
the glycerol-3-phosphate acyltransferase is a soluble enzyme that, unlike the E. coli
enzyme, shows preference for 18 : 1-ACP over 16 : 0-ACE The lysophosphatidic acid
acyltransferase, which is a component of the inner envelope of plastids, is extremely
selective for 16: 0-ACE The presence of a 16-carbon fatty acid at the 2-position is
therefore considered diagnostic for lipids synthesized in the plastids.
Relative fluxes through the prokaryotic and eukaryotic pathways vary between organ-
isms and among tissues. Plastids have the potential to use phosphatidic acid from the
prokaryotic pathway for all of their glycerolipid syntheses. However, not all plants do
so; in some cases, the prokaryotic acyl chain arrangement is found only in plastidial
phosphatidylglycerol, whereas galactolipids are derived from diacylglycerol imported
to the plastids from the ER. As indicated above, the eukaryotic acyltransferases of
the endoplasmic reticulum produce substantially more 18/18 than 16/18 lipids, and
it is chiefly the 18/18 units that are assembled into galactolipids by phmts with a
minor prokaryotic pathway. Because galactolipids become highly unsaturated, plants
that import diacylglycerol for galactolipids are rich in 18 : 3 and are called 18 : 3 plants.
Species in which most galactolipid is derived from the prokaryotic 18/16 or 16/16
diacylglycerol contain substantial 16 : 3 and are known as 16 : 3 plants.
Kunst et al. [10] have demonstrated that a 16 : 3 plant, Arabidopsis thaliana, may be
converted to a de facto 18 : 3 plant by a single mutation in plastidial glycerol-3-phosphate
acyltransferase. Under these conditions, 16 : 3 content is reduced dramatically, and when
isolated chloroplasts are labeled with glycerol-3-phosphate, only phosphatidylglycerol
is labeled. Nevertheless, the percentage of galactolipids in mutant plants is practically
identical to that in wild-type plants, emphasizing the ability of plants to compensate
for reduction of the prokaryotic pathway. Other studies in mutants have confirmed that
plants have an amazing capacity to adapt to many, but not all, perturbations of lipid
metabolism (Section 11).
6. Glycerolipid synthesis p a t h w a y s
A plant stores reserve material in its seeds in order to allow seedling growth of the next
generation until photosynthetic capacity can be established. The three major storage
materials are oil, protein and carbohydrate, and almost all seeds contain some of each.
However, their proportions vary greatly. For example, the amount of oil in different
species may range from as little as 1-2% in grasses such as wheat, to as much as 60%
of the total dry weight of the castor seed. With the exception of the jojoba plant, which
accumulates wax esters in seeds, plants store oil as triacylglycerol (TAG).
In the mature seed, TAG is stored in densely packed lipid bodies, which are roughly
spherical in shape with an average diameter of 1 g m (Fig. 3) [13]. This size does
not change during seed development, and accumulation of oil is accompanied by an
increase in the number of lipid bodies. The very large number of lipid bodies in an
oilseed cell (often >1000) contrasts strikingly with animal adipose tissue where oil
droplets produced in the cytosol can coalesce into a few or only one droplet. The
plant lipid bodies appear to be surrounded by a lipid monolayer in which the polar
headgroups face the cytosol, while the non-polar acyl groups are associated with the
non-polar TAG within. The membranes of isolated lipid bodies, which comprise less
than 5% of a lipid body's weight, contain both phospholipids and characteristic proteins
known as caleosins and oleosins. The recently discovered caleosins are calcium binding
proteins whose function is still unknown. Oleosins are small (15-26 kDa) proteins
that are believed to preserve individual lipid bodies as discrete entities. Desiccated
seeds lacking oleosins undergo lipid body fusion and cell disruption when rehydrated
(Leprince, 1998). The cDNAs encoding many oleosins have been cloned and each has a
sequence encoding a totally hydrophobic domain of 68-74 amino acids which is likely
to be the longest hydrophobic sequence found in any organism. Structurally, oleosins are
roughly analogous to the animal apolipoproteins which coat the surface of lipid droplets
during their transport between tissues.
When a seed germinates, the TAG stored in the lipid bodies becomes the substrate
for lipases. In at least some cases, peroxidation of polyunsaturated fatty acids by a lipid
106
Fig. 3. Thin-sectional view of cells in a cotyledon of a developing cotton embryo harvested 42 days after
anthesis. The cells are densely packed with lipid bodies and several large storage protein bodies (dark).
Magnification x9000. Photo courtesy of Richard Trelease, Arizona State University.
body lipoxygenase precedes the release of fatty acids from TAG [14]. Typically lipases
and lipid body lipoxygenase are active only after germination is triggered by imbibition
and other environmental signals. Fatty acids released by the lipid bodies are further
metabolized through the 13-oxidation pathway and glyoxylate cycle in the glyoxysomes
(Section 2.4).
4 amino acid changes have been shown to convert a desaturase into a hydroxylase
[16].
The reason for the great diversity in plant storage oils is unknown. However, the
special physical or chemical properties of the 'unusual' plant fatty acids have been
exploited for centuries. In fact, approximately one-third of all vegetable oil is used for
non-food purposes (Table 3). Reading the ingredients of a soap or shampoo container
reveals one of the major end uses of high lauric acid specialty plant oils. Other major
applications include the use of erucic acid (22 : 1) derivatives to provide lubricants and
as a coating for plastic films. Hydroxy fatty acids from the castor bean have over 100
industrial applications including plastic and lubricant manufacture. As discussed further
below, the ability of genetic engineering to transfer genes for some unusual fatty acid
production from exotic wild species to high yielding oil crops is now providing the
ability to produce new renewable agricultural products and to replace feedstocks derived
from petroleum.
As in animal tissues, it has been suggested that TAGs are produced by a relatively simple
four reaction pathway. According to this model, phosphatidic acid is synthesized by the
extraplastidial pathway (Section 5) and dephosphorylated to diacylglycerol. A third fatty
acid is then transferred from CoA to the vacant third hydroxyl of the diacylglycerol,
producing TAG. This last and single committed step is catalyzed by diacylglycerol
acyltransferase (Fig. 4, reaction 6). Although plants possess all of the enzymes for the
reactions above, the assembly of three fatty acids onto a glycerol backbone is not always
108
CDP-
18:1 choline CMP
~ 18:1
x_Z
3 E
18:1
18:1
acyl group I- 18:1
modification |lTZ~ ~
(~- choline 4 >
^ /CMP~
..... \
W~1" chline %CDP- \
2t
r /
El::
~-~ ""ch~ine18:1F
~" E~18:1 17
E2
it
OH
Fig. 4. Pathway depicting how flux through phosphatidylcholine (product of reaction 3) can promote acyl
group diversity in plant triacylglycerols. Production of18:2 (boxed) at the sn-2 position and its transfer
to TAG is used as a sample modification. Other fatty acid alterations may be substituted. Enzymes: I
= glycerol-3-phosphate:acyl-CoA acyltransferase: 2 = lysophosphatidic acid:acyl-CoA acyltransferase;
3 -- CTP:phosphatidate cytidylyltransferase; 4 = T6 18:l-desaturase or other fatty acid modifying
enzyme: 5 = phospholipid:diacylglycerol acyltransferase; 6 = diacylglycerol acyhransferase; 7 = acyl-
CoA : phosphatidylcholine acyltransferase or phospholipase plus acyl-CoA synthetase.
Although the basic reactions of TAG biosynthesis have been determined, several
fundamental and potentially related questions persist. As highlighted above, TAG and
membrane lipids frequently have radically different fatty acid compositions. How do
109
plants control which fatty acids are stored in TAG as opposed to which fatty acids are
restricted to membranes? Are unusual fatty acids excluded from membranes because
their physical and chemical idiosyncracies would perturb membrane fluidity or other
physical characteristics? Is TAG synthesis spatially distinct from the synthesis of
membrane lipids, or do enzyme specificities dictate the partitioning of fatty acid species
among glycerolipids? Although all of these factors may be significant, selectivity by
enzymes such as phospholipid:diacylglycerol acyltransferase for unusual fatty acids,
and editing of unusual fatty acids from phospholipids, are currently the best documented
[17,18].
8. Protective lipids
In plants, tissues are protected against desiccation and pathogens by both a cuticle and
epicuticular wax. The cuticle itself contains some wax, but is anchored to the plant
cell wall by cutin, a complex polyester of fatty acid derivatives with a wide range of
oxygen-containing functional groups (Fig. 5). 16- and 18-carbon dicarboxylic acids with
one or more hydroxyl groups are particularly common [19]. Arabidopsis transformed
with cutinase from a fungal plant pathogen not only develops a leaky cuticle, but also
suffers from fusions between leaves and flower parts (Sieber, 2000).
Surface waxes are complex mixtures including a range of very long-chain alkanes,
aldehydes and ketones as well as wax esters and their building blocks. Although only
one mutation in cutin formation has been identified (Wellesen, 2001), visual screening
of plant surfaces has allowed isolation of several wax mutants blocked in malonyl-CoA-
dependent elongation of fatty acids, decarbonylation and reduction (Post-Beittanmiller,
1996). The cDNA of an elongase required for extension of wax acyl units beyond 24
carbons has been cloned, and resembles the condensing enzymes involved in synthesis of
erucic acid in seed oil and wax ester precursors in jojoba seeds (Millar, 1999). A cDNA
encoding the wax synthase of jojoba seeds has been cloned and successfully expressed
in Arabidopsis (Lardizabal, 2000), and may provide clues to the corresponding genes
for surface waxes.
Bark, wound callus, and specialized tissues such as the endodermis that controls
entry into the root vascular system, have walls lined with suberin. Suberin, like cutin,
is a polyester incorporating fatty acids enriched in carboxyl and hydroxyl groups. In
addition to placement on the inner surface of cell walls rather than outside, the tough,
waterproof suberin differs from cutin in its preference for longer fatty acids and in its
incorporation of large amounts of phenylpropanoids [19].
! l O/~oC~Nc15H31
~
/ 0 ~ c%o
O ' # ' C ~ O~C~(CH2)8~C/(CH2)s~O~
~H
n
Fig. 5. Model showing some of the linkages in cuzin, after Kolattukudy [19]. Note the cross-linking made
possible by mono- and dihydroxy-fattyacids.
all belong to this group. Given that vital plant hormones such as gibberellin and
abscisic acid, plus many defensive compounds, are isoprenoids, the early steps of
this pathway have been studied intensely. However, surprisingly, it was not until
the late 1990s that researchers realized that plants have two very different pathways
for production of isopentenyl pyrophosphate, the five-carbon central precursor of all
isoprenoids [20]. For several decades it was known that, as in other organisms, plants
join three molecules of acetyl-CoA to form hydroxymethylglutaryl-CoA followed by
the highly regulated reduction of that compound to mevalonic acid. Furthermore,
plants contain multiple well-studied hydroxymethylglutaryl-CoA reductase genes that
111
are differentially expressed during development and in response to such stimuli as light,
wounding and infection. It was incorrectly suspected that this 'mevalonate' pathway
was localized in both cytosol and plastids and produced all classes of isoprenoids. The
story has now been clarified [20] with the discovery that plastids produce isopentenyl
pyrophosphate by a 'non-mevalonate' pathway that begins with the condensation
of pyruvate with glyceraldehyde-3 phosphate to produce 1-deoxy-D-xylulose-5-R At
least three additional enzymes are required to produce isopentenyl pyrophosphate in
the plastids. In parallel to work in plants, this pathway has also been demonstrated in
bacteria and algae. The non-mevalonate pathway in plastids is responsible for production
of the classic plant photosynthetic isoprenoids such as phytols and carotenoids, as well
as mono- and diterpenes.
The mevalonate pathway in the cytosol is responsible for biosynthesis of sterols,
sesquiterpenes and triterpenoids. After conversion of mevalonic acid to isopentenyl
pyrophosphate, three C5 units can be joined head to tail to produce a C15 compound,
farnesyl pyrophosphate. Two farnesyl pyrophosphates are then united head to head to
form squalene, the progenitor of the C30 isoprenoids from which sterols are derived. The
plant squalene synthetase, like its mammalian homologue, is found in the endoplasmic
reticulum and the reaction proceeds via a presqualene pyrophosphate intermediate. In
the last step prior to cyclization, squalene is converted to squalene 2,3-epoxide.
It is also in the cyclization step that photosynthetic and non-photosynthetic organisms
diverge. Whereas animals and fungi produce lanosterol, organisms with a photosynthetic
heritage produce cycloartenol. Despite the differences in the cyclization product, there
is substantial conservation between the enzymes responsible, with 34% identity between
an Arabidopsis cycloartenol synthase and lanosterol synthase.
A complex series of reactions including opening of the cyclopropane ring, double
bond formation and isomerization, demethylation of ring carbons, and methylation of
the side chain result in formation of a number of different plant sterols. Sitosterol
is the most common plant sterol (Fig. 6); however, plants normally contain mixtures
of sterols whose proportions differ from tissue to tissue. In addition, sterol esters,
sterol glycosides, and acylated sterol glycosides are common plant constituents whose
physiological significance is under scrutiny. Both cold adaptation and pathogenesis
drastically alter free and derivatized sterol pools. Plants also produce a steroid hormone,
brassinolide, required for both light-induced development and fertility. Interestingly, the
gene for a 5c~-reductase in the brassinolide pathway can complement the corresponding
reductase in the testosterone pathway [21 ].
Sphingolipids are usually considered minor constituents of plant lipids, accounting
for 5% or less of most lipid extracts. This fact, and the more complex methods needed
for their identification and characterization have resulted in a comparative lack of
information on plant sphingolipid biosynthesis and function. Nevertheless, sphingo-
lipids make up a substantial proportion (25% or more) of the composition of plasma
and tonoplast membranes, with the glucosylceramides constituting the largest fraction.
In addition, difficulties in extraction and analysis may have led to underestimates of
sphingolipid contents. As in animals (Chapter 14), sphingolipid biosynthesis begins
with condensation of palmitoyl-CoA with serine to form 3-keto-sphinganine, with the
enzyme from A. thaliana showing 68% similarity to the human homologue (Tamura,
112
C22 C24
Fig. 6. Sitosterol (24c~-ethylcholesterol),shown here, is the most common plant sterol, but plants generally
contain complex mixtures of sterols. Other prominent phytosterols differ from sitosterol as follows.
Campesterol, 24c~-methyl;stigmasterol, C22 double bond; dihydrospinasterol, move double bond from C5
to C7; spinasterol, move C5 double bond to C7, add C22 double bond; dihydrobrassicasterol, 24~-methyl;
brassicasterol, 2413-methyl,add C22 double bond.
OHC'~/'~v/~v.~COOH
~~x~,C/~OOH traumatin
,8:3 l
OHC~~~/COOH
t
OOH
12-oxo-cis-dodecenoic acid
~COOH cis-3-hexenal~ C H O
13-hydroperoxy-18:3
HO O
OH 12,13-ketol
O OH
12,13-epoxy-18:3
~'~V~~ ~~jCOOH
O
10 9,12-ketol
~ C O O H
12-oxo-phytodienoicacid
jasmonicacid
Fig. 7. Metabolism of 18:3 to oxylipins, l, lipoxygenase; 2, allenoxide synthase; 3, allene oxide cyclase; 4,
12-oxo-phytodienoicacid reductase, [3-oxidation;5, hydroperoxide lyase.
be further metabolized to 7-iso-jasmonic acid. In the last few years, it has become
clear that jasmonate is a key component of a wound-signaling pathway that allows
plants to protect themselves against insect attack. When experimentally applied to
114
11.1. Mutants in lipid metabolism have helped link lipid structure and function
Two other major benefits have been derived from the Arabidopsis lipid mutants. First,
the physiological effects of the mutations have provided the opportunity to evaluate the
relationships between lipid structure and function. There has been a long-term assump-
tion, based on the strong association of high levels of polyunsaturated fatty acids with
photosynthetic membranes and the conservation of this property among higher and lower
plant species, that these fatty acids must be essential to photosynthesis. However, many
attempts to understand the relationships between membrane fatty acid composition and
cell physiology or photosynthesis have led to equivocal results. The isolation of mutants
totally lacking certain unsaturated fatty acids has now provided much more convincing
evaluations of their function and indeed, the results have forced re-evaluation of several
previous hypotheses. For example, A3-trans-hexadecenoate is an unusual plant fatty acid
which is associated with phosphatidylglycerol of chloroplast membranes, is evolutionar-
ily conserved, and is synthesized in coordination with the assembly of the photosynthetic
apparatus. These observations led to the suggestion that A3-trans-hexadecenoate is a
highly essential component of photosynthesis. However, mutants which contain no
detectable A~-trans-hexadecenoate grow as well as wild type plants, and all photo-
synthetic parameters examined appear normal (Browse, 1985). A minor difference in
stability of some components of the photosystem can be detected by polyacrylamide
115
gel electrophoresis. It has been concluded from such analyses that, although A~-trans -
hexadecenoate may facilitate assembly of the light harvesting complex into thylakoids, a
more obvious phenotype could be restricted to certain unusual environmental conditions.
As mentioned above, a number of mutants blocked in the production of polyunsat-
urated fatty acid biosynthesis have also been isolated (Table 4). Because leaves have
desaturases both in chloroplasts and in the endoplasmic reticulum, single mutations
lead only to partial reduction of polyunsaturated fatty acid levels. Again, these mutants
grow normally under most conditions and have normal photosynthetic parameters. How-
ever, several alterations in physiology are observed including changes in chloroplast
ultrastructure, a reduction in the cross-sectional area of chloroplasts, and increased sta-
bility to thermal disruption of photosynthesis. Moreover, whereas wild-type Arabidopsis
plants are chilling resistant and can reproduce normally at temperatures as low as 6C,
the mutants blocked in plastidial A 7 (f~ld5) and co6 ~ d 6 ) desaturation become chlorotic
at 6C and show a 20-30% reduction in growth rate relative to the wild-type plant.
The fad2 mutants, in which the endoplasmic reticulum o)-6 desaturase is blocked, are
even more sensitive to 6C and die if left at this temperature for several days. These
results demonstrate that polyunsaturated fatty acids are essential fbr maintaining cellular
function and plant viability at low temperatures.
While most mutants which are reduced only in polyunsaturated fatty acid synthesis
grow and develop normally at 22C, a high-stearate mutant with 14% 18:0 is strikingly
abnormal (Fig. 8) [26]. Many cell types fail to expand, resulting in mutant plants
growing to less than one-tenth the size of wild-type. At higher growth temperatures
(36C), the effects are less dramatic, suggesting that the physical properties or fluidity
of highly saturated membranes are less impaired under these conditions.
Other large scale alterations in membrane fatty acid composition and phenotypes
have been obtained by creation of multiple-mutant lines. When mutants defective in
endoplasmic reticulum co6-desaturase were crossed with plants defective in the plastid
co6-desaturase, double mutants could be recovered only on sucrose-supplemented media.
The sucrose grown plants, which contained less than 6% polyunsaturated fatty acids,
were chlorotic and unable to carry out photosynthesis but otherwise remarkably normal.
These results, while confirming the significance of polyunsaturated fatty acids to
photosynthesis, indicate that the vast majority of membrane functions can proceed
despite drastically reduced levels of polyunsaturates [27].
Triunsaturated fatty acids normally dominate chloroplast membranes and thus are the
most abundant fatty acids on the planet. By constructing a triple mutant of fad3, fad7
and fad8, it has been possible to eliminate triunsaturated fatty acids from Arabidopsis
without affecting 16:2 and 18 : 2 production (McConn, 1996). Surprisingly, these plants
are able to grow, photosynthesize, and even flower. However, they are male sterile and
therefore cannot produce seeds. This observation led to the discovery of a very different
role for jasmonate. This mutant cannot synthesize jasmonate because it lacks the 18 : 3
precursor, and the plants are male-sterile because pollen does not mature properly and is
not released from the anthers. Application of jasmonate or linolenic acid to the anthers
restores fertility, demonstrating that jasmonate is a key signal in pollen development.
This result is a dramatic example of a change in fatty acid composition having a very
specific effect on an essential developmental and tissue specific reproductive process.
116
.~'~ _, .~~8= a
" o~ rj
o ~
-t
.&
-..!
.b. r=
~ < .2
~.-~
--> ~
o
E
t-,r.,
8
"Fo ~- ~ _~ ~ ~:
o
- - v v ;~ :~ ,,--, ~o
"6
e-,
.=_
,-j
-8
oo
F~
117
Fig. 8. Increased stearic acid causes severe dwarfing of Arabidopsis. A wild-type Arabidopsis plant (left) is
compared to the.lab2 mutant (right) in which leaf stearic acid content has increased from 1 to 14%. Photo
courtesy of John Browse, Washington State University.
A. thaliana mutants have also provided a means of cloning genes difficult to isolate by
other methods. As in other kingdoms, the membrane bound enzymes of plants have been
notoriously difficult to purify and characterize. However, cDNAs or genes encoding a
number of these enzymes have now been isolated using molecular genetic strategies
based on mutations. Several approaches have been successful. A cDNA encoding the
co-3 desaturase which converts linoleic to linolenic acid was isolated in 1992 by Arondel
et al. [28] after a mutation leading to the loss of function was genetically mapped and
a yeast artificial clone corresponding to this region was selected. The genomic region
was then used to screen a cDNA library, and some of the clones which hybridized to
the yeast artificial chromosome had sequence similarity to cyanobacterial desaturases.
These clones subsequently were shown to complement the loss of 18 : 3.
Gene 'tagging' strategies have also proved enormously valuable in identifying
clones of membrane bound enzymes. Arabidopsis genes can be 'tagged' by insertional
118
In recent years, tremendous progress has occurred not only in the isolation of many
plant lipid biosynthetic genes, but also in the use of these genes to manipulate plant oil
composition. As shown in Table 5, both substantial changes in seed oil composition and
introduction of unusual fatty acids to heterologous species have been achieved. Progress
in this area has been accelerated by several industrial laboratories whose goal has been
the production of higher value oilseeds.
Table 5
Some examples of genetic engineering of plant lipid metabolism
Soybean
Corn sz D
Sunflower
Canola
Olive
Palm
Beef tallow
0 10 20 30 40 50 60 70 80 90 100
Fig. 9. Fatty acid composition of dietary vegetableoils and beef tallow. The values shown represent typical
compositions of varieties grown commercially. Lines modified substantially through breeding or genetic
engineering are available for soybean, canola, corn and sunflower.
and 7-1inolenic acid (18 : 3-A6'~'12), which is implicated in relieving arthritis and other
conditions [30]. y-Linolenic acid is produced in some fungi and the seeds of some
plants by the desaturation of linoleic acid to 7-1inolenic acid by an endoplasmic
reticulum-localized A6-desaturase. Identification of cDNAs encoding this desaturase
from borage and the fungus Mortierella alpina has led to their heterologous expression
in plants. Expression of the Mortierella gene in Brassica napus seed resulted in 47%
7-1inolenic acid production (Ursin, 2000). Expression of the fungal A6-desaturase in a
low e~-linolenic acid canola line further enhanced 7-1inolenic acid production to 68%.
When canola lines with high c~-linolenic content were crossed with high 7-1inolenic
acid lines, up to 17% stearidonic acid was produced. These examples illustrate that the
level of desired end-product can sometimes be substantially increased, although often
unpredictably, by the choice of host and/or enzyme source.
is also a solid at room temperatures and therefore ideal for margarine and shortening
manufacture. Although petroselinic acid is a major component of seed oils in species
such as coriander and carrot, these crops have a low yield of oil per hectare. It is
therefore hoped that an oilseed crop can be engineered to become a commercially viable
source of petroselinic acid. When a cDNA encoding the acyl-ACP desaturase involved
in petroselinic acid synthesis was cloned from coriander and used to transform other
plants, petroselinic acid was produced but only at low levels. Therefore, to achieve
economic production of petroselinic acid, it may be necessary to introduce additional
genes. It is now known that coriander and its relatives possess several proteins devoted
to production of this special fatty acid, including ACP isoforms, a modified condensing
enzyme (3-ketoacyl-ACP synthase) specific for the elongation of cis-4-16: 1-ACP to
cis-6-18 : I-ACP and a novel acyl-ACP thioesterase specific for petroselinoyl-ACR
As described above, plants have evolved the ability to produce a diverse range of
fatty acid structures. A number of specialty fatty acids have already been extensively
exploited for industrial uses such as lubricants, plasticizers and surfactants (Table 3). In
fact, approximately one-third of all vegetable oil is now used for non-food products, and
this figure is expected to increase as petroleum reserves are depleted. Thus, in addition
to providing food, oilseed crops can be considered efficient, low polluting chemical
factories which are able to harness energy from sunlight and transform it into a variety
of valuable chemical structures with a multitude of non-food uses.
Pathway
4:0-ACP
Lauric Acid
12:0 12:0 I 4MCTE 12:0-ACP
14:0 14:0-ACP
"o
'~ 16:0 16:0-ACP
<
18:0-~ACP
~ 18:1
u.
18:1-~ACP
18:2
Wildtype 1
[] Transgenic J
18:3
I J
10 20 30 40 50 60
Mole %
Fig. 10. Genetic engineering of rapeseed oil to produce laurie acid. tool% of major fatty acids in a typical
canola cultivar are compared to the composition achieved through genetic engineering using a California
Bay medium chain acyl-ACP thioesterase (MCTE) under control of a napin promoter.
It is now possible to say that the biosynthetic pathways have been determined for all
major plant lipids and most of the genes identified for enzymes in these pathways have
been cloned. Clearly there has been great progress, although the biosynthetic pathways
of sphingolipids, trans-A'~-16 : 1 and some details of several pathways remain elusive.
In addition, the enzymes involved in the production of many unusual fatty acids found
in seed oils are largely unexplored.
One area of expanding interest is the production of lipid hormones and signal
molecules. Several lipids including phosphatidylinositol phosphates, diacylglycerol and
N-acylphosphatidylethanolamine have been implicated in signal transduction in plants.
Another intriguing similarity between plants and animals is their use of oxygenated
fatty acids in response to wounding. As mentioned in Section 10, jasmonate, a plant
growth regulator derived from 18:3, is able at femtomolar concentrations to induce
proteinase inhibitors and other plant defense genes. Like leukotriene synthesis in
animals, jasmonate biosynthesis begins with the generation of a hydroperoxide by
lipoxygenase. Jasmonate itself contains a cyclopentane ring comparable to those of
prostaglandins. The common roles and origins of oxygenated fatty acids in plants and
animals suggest a very early common ancestor for these pathways.
Application of molecular genetics and genomics to problems in lipid biochemistry
should continue to expand. Particularly stimulating has been the complete sequenc-
ing of the Arabidopsis genome and the availability of over 1 million expressed
sequence tag (EST) sequences from a variety of plants. A recent survey of Ara-
bidopsis genes involved in plant acyl lipid metabolism identified over 450 genes
124
Table 6
Intemet resources related to plant lipid metabolism
Organization Web site content URL
National Plant Lipid Directory of scientists http://www.msu.eduluser/ohhogge/
Consortium (NPLC) involved in plant lipid
research.
E-mail newsgroup tier
int~rmation on plant lipids.
Abstracts of NPLC meetings
Michigan State University Survey and catalog of genes http://www.canr.msu.edu/lgc/index.html
for plant lipid metabolism. http://www.plantbiology.msu.edu/
gene_survey/fron| page.htm
Gene expression profiles http://www.bppmsu.edu/Seed/SeedArray.htm
based on plant lipid ESTs
and microarrays
Kathy Schmid, Butler Links to many oilseed http:lYolue.butler.edu/~kschmidllipids.html
University research laboratories and
web sites
Benning and Ohlrogge Database and analysis of http://benningnt.bchmsu.edu
Laboratories > 10,000 ESTs from
developing ArabidopsL~
seeds
USDA Oilseed Database Data on fatty acid www.ncaur.usda.gov/nc/ncdb/search.html-ssi
composition of seeds of
thousands of species
of how (and when) they fit together that defines the challenge. Microarrays that permit
simultaneous monitoring of expression of many genes have begun to provide a more
global overview of how genes work together to control seed metabolism (Girke, 2000).
Together with the ability to rapidly over- and under-express genes in transgenic plants
and the strengths of classical biochemistry, such recent advances in analytical techniques
should allow us to enter a new stage of lipid research emphasizing the interplay between
metabolic compartments and the control of lipid synthesis during the plant life cycle.
Abbreviations
References
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154
O O O O OH O O H3C OH O O
dxs dxr ygbP
O P H3C P HO P
H3C + H O OH O OH O OH
O OH O OH NADPH NADP+ OH CTP PPi
1 2 3 4
NH2 NH2
O
N OH N O O
O P
H3C O P O O
H3C OH O O O O O N ychB H3C O O O O O O N ygbB
HO P
HO P P O HO P P O O O
O O O O O O - CDP
H H OH
OH ATP ADP OH
HO OH HO OH
5 6 7
CH3 O O O O
P P
H2C O O OH
CH3 O O O O idi
gcpE lytB
HO P P
O O OH
8 CH3 O O O O
P P
H3C O O OH
10
in Streptomyces hygroscopicus and S spheroides, respectively containing the three known genes of the MEP pathway (dxs,
[20]. DXP is not only an isoprenoid precursor, but was dxr and ygbP, Figure 1). They encode the enzymes
previously known as a precursor for thiamine diphosphate responsible for the further activation of ME:
and pyridoxol phosphate in E coli. The search for E coli phosphorylation of the C(2)-hydroxy group of 4-
mutants auxotrophic to ME led to the identification of the diphosphocytidyl ME (ychB) [26] and the conversion of the
DXP reductoisomerase gene and to the characterization of former intermediate, 4-diphosphocytidyl ME 2-phosphate
the corresponding enzyme [21,22]. This enzyme requires a (6; Figure 1) into ME cyclodiphosphate (7; Figure 1) (yfgB)
2+ 2+ 2+
divalent cation (Mg , Mn or Co ). It catalyzes the [27]. Deletion of the ygbP, ychB or ygbB gene in E coli was
rearrangement of DXP into methylerythrose phosphate and rescued by the insertion of the three genes encoding the
the concomitant NADPH-dependent reduction of the enzymes permitting the utilization of exogenous MVA
aldehyde into 2-C-methyl-D-erythritol 4-phosphate (MEP) (4; (MVA kinase, phospho-MVA kinase and diphospho-MVA
Figure 1) [23,24]. MEP shows the typical branched C(5)- decarboxylase). This confirmed that they are essential for
isoprene skeleton. It can be considered as the first isoprenoid biosynthesis [28-30].
hemiterpene of the pathway. As no other role is known for
2 2
MEP, the DXP reductoisomerase most probably catalyzes Incorporation of [4- H]DX or [3- H]ME into the prenyl
the first committed step of the pathway, which is named chains of ubiquinone and menaquinone of E coli resulted in
3
after the substrate of this enzyme. Incubation of [ H]-labeled a different labeling pattern in IPP and DMAPP; deuterium
MEP with crude cell-free extracts from E coli in the presence retention in the isoprene units derived from DMAPP and
of nucleotide triphosphates resulted in the formation of an complete loss in those derived from IPP [31,32]. In
adduct. A small gene cluster encoding the genes of the next addition, the IPP isomerase activity is barely detectable in
three enzymes was found when searching for a gene this bacterium, and the deletion of the IPP isomerase gene,
encoding an enzyme using a polyol phosphate (like MEP) the enzyme ensuring the interconversion of IPP (9; Figure 1)
and a nucleotide triphosphate. This resulted in the finding of and DMAPP (10; Figure 1), is not lethal for this bacterium
the homologies between the diphosphocytidine ribityl [33]. This suggested the presence of a branching in the
synthase and the unannotated ygbP gene from E coli [25]. MEP pathway, allowing a separate synthesis of IPP and
Cloning of this gene and overexpression of the DMAPP. Definitive proof for such a branching was
corresponding protein allowed the identification of the 4- identified by genetic methods [34]. An engineered E coli
diphosphocytidyl ME (5; Figure 1) synthase. The next two strain lacking the DXP reductoisomerase and capable of
genes of this cluster were always found in genomes already utilizing MVA grew normally either in the presence of ME
156 Current Opinion in Investigational Drugs 2004 Vol 5 No 2
or MVA. In growth conditions on MVA, only IPP is Table 1. Distribution of the MEP pathway.
synthesized, and cells have to rely on an IPP isomerase for Pathway Reference
the synthesis of DMAPP. After additional deletion of the MEP
IPP isomerase idi gene, MVA no longer supported growth, Acinetobacter calcoaceticus [90]
Actinobacillus actinomycetemcomitans [48]
indicating that there was no other way to interconvert IPP
Actinobacillus pleuropneumoniae [50]
and DMAPP than via the dimethylallyl diphosphate Actinoplanes sp A40644 [48]
isomerases (IDI) enzyme. However, the cells grew Alicyclobacillus acidoterrestris [7]
normally on free ME, demonstrating that IPP and DMAPP Anaplasma phagocytophilum] [50
are independently synthesized in the MEP pathway from Aquifex aeolicus [48]
an ME derivative. Bacillus anthracis [50]
Bacillus cereus [50]
A bioinformatic search in fully sequenced bacterial genomes Bacillus subtilis [48]
for genes accompanying the known genes of the MEP Bacteroides fragilis [50]
pathway led to the identification of two additional genes, Bacteroides thetaiotaomicron [50]
Bifidobacterium longum [50]
gcpE [35,36] and lytB [37,38]. These genes are essential in the
Bordetella bronchiseptica [50]
MEP pathway. Their deletion in E coli was rescued by the Bordetella pertussis [48]
addition of mevalonate to the culture medium after Brucella melitensis [50]
introducing the genes, allowing the utilization of this Brucella suis [50]
unnatural substrate for E coli [35,36,38]. The Burkholderia caryophylli [90]
hydroxymethylbutenyl 4-diphosphate synthase (GcpE) Burkholderia cepacea [50]
enzyme converts ME cyclodiphosphate into (E)-4-hydroxy- Burkholderia gladioli [90]
3-methylbut-2-enyl diphosphate (HMBPP) (8; Figure 1) Burkholderia mallei [50]
[39,40] by two successive one-electron transfers induced via Burkholderia pseudomallei] [50]
a [4Fe-4S] cluster [41,42]. The LytB enzyme catalyzes the Campylobacter jejuni] [48]
last step of the pathway, converting HMBPP into IPP or Caulobacter crescentus [48]
Chlamydia muridarum [49]
DMAPP and being responsible for the branching in the
Chlamydia pneumoniae [48]
pathway [43-45]. This last step involves, like the GcpE-
Chlamydia trachomatis [48]
catalyzed reaction, two one-electron transfers via a [4Fe-4S] Chlamydophila pneumoniae] [50
cluster [46,47], which has been directly characterized by Chlorobium tepidum [48]
electron paramagnetic resonance spectroscopy [47]. Citrobacter freundii [90]
Clostridium acetobutylicum [48]
Most steps of the MEP pathway described above for E coli Clostridium botulinum [50]
were also characterized in plants [11], where this pathway Clostridium difficile [48]
is localized in chloroplasts as shown by the additional Clostridium perfringens [50]
Clostridium tetani [50]
putative plastid-targeted N-terminal sequence found in all
Corynebacterium ammoniagenes [48]
enzymes. Corynebacterium diphtheriae [48]
Deinococcus radiodurans [48]
Distribution of the MEP and MVA pathways: Ehrlichia chaffeensis [50]
The MEP pathway, a target for antibacterial Erwinia carotovora [90]
Escherichia coli [7,48]
and antiparasitic drugs Eubacterium sp [50]
First insight into the distribution of the MEP pathway has
Francisella tularensis [50]
been demonstrated biochemically, usually after incubation Fusobacterium nucleatum [50]
13
of [ C]-labeled substrates (eg, glucose) and analyzing the Gardnerella vaginalis [50]
labeling pattern of the isoprenoids by NMR spectroscopy. Haemophilus ducreyi [50]
This approach was first applied to bacteria, and later Haemophilus influenzae [48]
extended to unicellular algae, liverworts and plants [3,4,11]. Helicobacter jejuni [48]
In unicellular green algae, it is the only pathway for Helicobacter pylori [50]
isoprenoid biosynthesis. In other investigated algae phyla Klebsiella pneumoniae [50]
and in plants, its presence is restricted to the chloroplast for Leptospira interogans [50]
the synthesis of the essential isoprenoids of the Listeria monocytogenes [50]
photosynthetic apparatus (phytol from chlorophylls, Mannheimia haemolytica [50]
Methylobacterium fujisawaense [7,48]
carotenoids and plastoquinone), of mono- and diterpenoids
Methylobacterium organophilum [6]
and isoprene. Sterols and triterpenes, most sesquiterpenes Moraxella catarrhalis [50]
and the prenyl chain of ubiquinones are synthesized via the Mycobacterium avium [48]
MVA pathway in the cytoplasm. Exchanges between the Mycobacterium bovis [48]
two cellular compartments have, however, been regularly Mycobacterium leprae [48]
reported and cross-talk between the two compartments is Mycobacterium phlei [90]
probably a way for the regulation of isoprenoid biosynthesis Mycobacterium smegmatis [90]
in plant cells [11]. Once the genes of the pathway were Mycobacterium tuberculosis [48]
known, a bioinformatic analysis of known genomes pointed Mycoplasma penetrans [50]
out the broad distribution of the genes of the MEP pathway Neisseria gonorrhoeae [48]
(Table 1). Neisseria meningitidis [48]
Isoprenoid biosynthesis as a novel target for antibacterial and antiparasitic drugs Rohmer et al 157
Table 1. Distribution of the MEP pathway (continued). Although the MVA pathway has been found in some
Pathway Reference bacteria, the MEP pathway represents probably the most
MEP widespread pathway for isoprenoid biosynthesis in
Nocardia brasiliensis [91] [91] eubacteria (Table 1). The known distribution of the two
Neorickettsia sennetsu [50] [50]
Pasteurella multocida [50] [50]
isoprenoid pathways merely reflects lateral gene transfer
Peptostreptococcus sp [50] rather than phylogenetic relationship [48,49,50]. The
Porphyromonas gingivalis [48] simultaneous presence of both pathways has been only
Prevotella intermedia [50] related in some Streptomyces species [51]. The MEP pathway
Proteus mirabilis [50] is utilized during the exponential growth phase for the
Providencia stuartii [50] biosynthesis of essential metabolites, such as the prenyl
Pseudomonas aeruginosa [90]
chains of the menaquinones, whereas the MVA route is
Pseudomonas fluorescens [90]
Pseudomonas putida [50] involved in the synthesis of the prenyl moiety of antibiotics
Psychrobacter sp [50] of mixed origin during the stationary phase. The MEP
Ralstonia pickettii [90] pathway is found in many pathogenic bacteria as well as in
Rhodobacter capsulatus [48] opportunistic pathogens (Table 1). It is also present in the
Rhodopseudomonas acidophila [6] parasitic Plasmodium species [52].
Rhodopseudomonas palustris [6]
Salmonella enterica [50]
In contrast, the MEP pathway is absent in animals, yeasts
Salmonella enteritidis [50]
Salmonella typhi [48]
and fungi. There is neither biochemical proof from labeling
Salmonella typhimurium [90] experiments for its presence, nor reports of the
Serratia marcescens [50] corresponding genes in the genomes of these organisms.
Shewanella putrefaciens [48] This makes the MEP metabolic route an interesting target for
Shigella flexneri [50] the design of novel antibacterial and antiparasitic drugs,
Shigella dysenteriae [50] with minimal side effects for the patient. Even if most
Sphingobacterium multivorum [92]
bacterial species do not produce large amounts of
Streptomyces aeriouvifer CL190* [48]
Streptomyces blastmyceticum
isoprenoids, some of these metabolites are essential for all
[48]
Streptomyces exfoliatus [48]
bacteria (eg, bactoprenol diphosphate, the carbohydrate
Streptomyces ghanaensis [48] carrier for the biosynthesis of peptidoglycan of the cell wall)
Streptomyces griseolosporeus* [48] or in most of them (prenyl chain of ubiquinones and
Streptomyces niveus [48] menaquinones from electron transport chains). Any
Streptomyces spheroides [48] inhibition of the biosynthesis of these essential terpenoids is
Streptomyces sp QC45B [93] lethal.
Streptomyces sp UC 5319 [94]
Streptomyces sp JP95 [95]
Synechocystis sp PCC 6803
Enzymes of the MEP pathway: New targets for
[48]
Tannerella forsythensis [50] antibacterial and antiparasitic drugs
Thermotoga maritima [48] Due to the relatively recent discovery of the MEP pathway,
Thiobacillus ferrooxidans [48] few investigations have been published on the potential role
Treponema denticola [50] of this metabolic route for the development of new
Tropheryma whipplei [50] antibacterial drugs.
Treponema pallidum [48]
Vibrio cholerae [48] The discovery of the genes implied in the MEP pathway
Vibrio vulnificus [50] made recombinant enzymes available, thus making the
Wolbachia sp [50] characterization of their catalytic properties possible. Kinetic
Xylella fastidiosa [49]
data have been obtained for the DXP synthase of Rhodobacter
Yersinia pestis [48]
capsulatus [18] and Streptomyces coelicolor [19], the DXP
Yersinia enterocolitica [50]
reductoisomerase from E coli [23,24] and Streptomyces
Zymomonas mobilis [7]
coelicolor [19], the 4-diphosphocytidyl-2-C-methylerythritol
MVA
Borrelia burgdorferi [48] synthase from S coelicolor [19] and from the higher plant
Chloroflexus aurantiacus [48] Arabidopsis thaliana [53], and the 2-C-methylerythritol 2,4-
Enterococcus faecalis [48] cyclodiphosphate synthase from Plasmodium falciparum [54].
Flavobacterium sp [96] Amino acid residues involved in the catalytic activity have
Lactobacillus helveticus [97] been characterized by directed mutagenesis for the DXP
Lactobacillus plantarum [48] synthase [55] and for the DXR [56] from E coli. X-ray
Myxococcus fulvus [48,90] structures are available for the DXP reductoisomerase
Nannocystis exedens [98] [57,58], the 4-diphosphocytidyl-2-C-methylerythritol
Paracoccus sp [99] synthase [59,60], the 4-cytidine 5'-diphospho-2-C-methyl-D-
Staphylococcus aureus [48]
erythritol kinase [61,62] and the 2-C-methylerythritol 2,4-
Staphylococcus carnosus [48]
cyclodiphosphate synthase [63-66] from E coli.
Streptococcus mutans [48]
Streptococcus pneumoniae [48]
Streptococcus pyogenes
Fosmidomycin (13; Figure 2) is an antibiotic, which was
[48]
discovered in the Fujisawa Research Laboratories [67]. It has a
*Bacteria possessing the MEP and the MVA pathways. potent antibacterial activity against many Gram-negative and
some Gram-positive bacteria, has a low toxicity for animals
158 Current Opinion in Investigational Drugs 2004 Vol 5 No 2
and is efficient against experimental infections in mice [68]. A parasitemia was lower than 1% at concentrations as low as 20
correlation was found in bacteria between fosmidomycin mg/kg of either drug [52]. The diaryl esters (15; Figure 2),
insusceptibility and the ability to incorporate MVA into prodrugs of FR-900098, demonstrated superior activity
isoprenoids. MVA incorporating bacteria such as Lactobacillus against P vinckei in mice after oral administration. One of
spp, Streptococcus spp and Arthrobacter spp were all these prodrugs even demonstrated a comparable activity by
fosmidomycin resistant, whereas Micrococcus spp, Bacillus spp oral or intraperitoneal administration [74]. Density Functional
and the tested Gram-negative bacteria (E coli, Enterococcus Theory (DFT) ab initio calculations were performed on DXP,
cloacae and Pseudomonas aeruginosa) were sensitive to the drug the substrate of the DXP reductoisomerase, as well as on its
[69]. In addition, fosmidomycin treatment was followed by a inhibitors fosmidomycin and FR-900098, in order to
decrease of the ubiquinone and menaquinone content in E determine their structures and charge distributions. This was
coli, and of carotenoids in Micrococcus luteus. These data intended for the further development of biologically active
suggested that fosmidomycin had an effect on isoprenoid inhibitors as well as parameters for docking experiments [75].
biosynthesis and, according to the above mentioned list of The crystal structure of the DXR complexing manganese and
tested bacteria, on an enzyme of the mevalonate-independent fosmidomycin showed that the inhibitor behaves like a DXP
MEP pathway. This was verified once the enzymes of the (3; Figure 1) analog. The oxygen atoms of the (N-formyl-N-
MEP pathway were available. Fosmidomycin was reported as hydroxy)amino group provide two ligands for the
a mixed type inhibitor of the DXP reductoisomerase from E coordination of manganese, and the phosphonate group
coli (Ki = 38 nM) [70] and as a competitive inhibitor of the linked through a three-methylene spacer is recognized by
Zymomonas mobilis enzyme (Ki = 0.6 M) [71]. It also inhibits hydrogen binding in a specific pocket [76]. Resistance to
the enzyme from higher plants [72,73]. In addition, the fosmidomycin was investigated in E coli [77]. Adenylate
recombinant DXP reductoisomerase from Plasmodium cyclase or glycerol 3-phosphate transport deficient mutants
falciparum is inhibited by fosmidomycin and the related were found to be resistant to fosmidomycin, indicating that
antibiotic FR-900098 (14; Figure 2) in a dose-dependent the antibiotic is transported via the glycerol 3-phosphate
manner [52]. An additional antibiotic related to transporter, the gene of which, like other genes involved in
fosmidomycin, FR-33289 (16; Figure 2) is known, but nothing glycerol metabolism, is highly dependent on the presence of
has been reported on its activity [70]. The presence of the cyclic AMP.
MEP pathway in Plasmodium spp is related to the apicoplasts,
which are plastid-like organelles acquired by the members of Little additional information is available on the inhibition of
the phylum Apicomplexa by endosymbiosis of an algal other enzymes of the MEP pathway. Fluoropyruvate (11;
ancestor. Fosmidomycin and FR-900098 are potent Figure 2) inhibits DXP synthase, as expected for a thiamine
antimalarial agents. For example, mice infected with diphosphate dependent enzyme, with IC50 values of 400 and
Plasmodium vinckei were treated intraperitoneally with 80 M for the Pseudomonas aeruginosa and E coli enzymes,
fosmidomycin (> 10 mg/kg) or FR-900098 (5 mg/kg) and respectively [78]. 5-Ketoclomazone (12; Figure 2), a
were apparently free of parasites, whereas untreated control breakdown product of the herbicide clomazone, inhibits, to
animals died from infection after 7 days. After oral some extent, the DXP synthase of the green alga
administration of either fosmidomycin or FR-900098 (50 or Chlamydomonas (IC50 ~ 0.1 mM), whereas clomazone itself
100 mg/kg), mice were seemingly free of parasites, and has no effect [79]. Free DX is readily incorporated into the
H3C O
O OH O O
H3C
F O H N P
N OH
O
O
F F O O
Cl
11 12 13
OH O O OH O O Ar OH OH O O
H3C N P H3C N P Ar H3C N P
OH O OH
O O O
14 FR-900098 15 16 FR-33289
OH F OH OH O O O O
H3C P H3C P
F OH F OH O OH O OH
O OH O OH O O OH
17 18 19 20
Isoprenoid biosynthesis as a novel target for antibacterial and antiparasitic drugs Rohmer et al 159
in Staphylococcus and Streptococcus spp: 7. Rohmer M, Knani M, Simonin P, Sutter B, Sahm H: Isoprenoid
biosynthesis in bacteria: A novel pathway for the early steps leading
Another target? to isopentenyl diphosphate. Biochem J (1993) 295(2):517-524.
Even in some bacteria possessing the MVA pathway, This is the first report of the existence of an alternative mevalonate-
isoprenoid biosynthesis may be a target for antibacterial independent pathway for isoprenoid biosynthesis in bacteria. The origin of the
drugs. A novel type II IPP isomerase has been characterized carbon atoms of isoprene units was determined, and the key rearrangement
step was characterized. This paper represents, with reference [9], the
in archaea and in some Gram-positive bacteria (eg, starting point of all further research on the novel metabolic pathway.
Streptomyces sp CL190, Staphylococcus aureus, Streptococcus
pneumoniae, Streptococcus pyogenes and Enterococcus faecalis) 8. Rohmer M, Seemann M, Horbach S, Bringer-Meyer S, Sahm H:
Glyceraldehyde 3-phosphate and pyruvate as precursors of
[85]. The enzyme is a flavoprotein that requires the isoprenic units in an alternative non-mevalonate pathway for
simultaneous presence of both flavin mononucleotide and terpenoid biosynthesis. J Am Chem Soc (1996) 118(11):2564-2566.
D-Glyceraldehyde phosphate and pyruvate are identified as the first
NADP to produce DMAPP from IPP and is characterized by precursors of the MEP pathway.
completely different catalytic properties and kinetic
parameters than those of all other known IPP isomerase, 9. Schwarz M: Terpen-biosynthese in Gingko biloba: Eine
berraschende geschichte. PhD Thesis Nb 10951, Eidgenssische
including the mammalian enzymes. Technische Hochschule, Zrich, Switzerland (1994).
In addition to reference [7], this thesis presents the first report on the
Conclusion occurrence of the mevalonate-independent route in higher plants. This
research was performed independently of the research on bacterial
The MEP pathway for isoprenoid biosynthesis is isoprenoids. See also reference [10].
widespread in bacteria. It has been characterized by
10. Schwarz M, Arigoni D: Gingkolide biosynthesis. In: Comprehensive
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absent in animals and humans, and the corresponding
12. Rohdich F, Kis K, Bacher A, Eisenreich W: The non-mevalonate
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fosmidomycin resistance has been characterized for E coli via isoprenoiden in Escherichia coli. II. Beitrag zur aufklrung der
a protein facilitating the efflux of the drug [87], this aspect biosynthese von vitamin B12 in Propionibacterium shermanii. PhD
Thesis Nb 10978, Eidgenssische Technische Hochschule, Zrich,
is particularly relevant in times where nosocomial diseases Switzerland (1994).
and resistance to antibiotics represent a severe problem. The This thesis presents the first direct evidence that a 1-deoxy-D-xylulose
first results on the efficacy of fosmidomycin against malaria derivative is the precursor of isoprene units in Escherichia coli. Incorporation
experiments using labeled deoxyxylulose were later a key tool for the
or urinary tract infections in adults indicate the strong and elucidation of the MEP pathway.
promising potentiality of the MEP pathway as a drug target
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therefore expected that in the near future, the other enzymes discovery of DXP reductoisomerase, followed by the characterization of the
will be investigated. role of fosmidomycin.
160 Current Opinion in Investigational Drugs 2004 Vol 5 No 2
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2
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36. Altincicek B, Kollas AK, Sanderbrand S, Wiesner J, Hintz M, Beck E,
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39. Hecht S, Eisenreich W, Adam P, Amslinger S, Kis K, Bacher A, Arigoni
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three additional steps downstream of the formation of MEP. See also 2-enyl diphosphate: Chemical synthesis and formation from
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Biosynthesis of terpenoids: YgbB protein converts 4- Rohmer M: Isoprenoid biosynthesis through the methylerythritol
diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate to 2C- phosphate pathway: The (E)-4-hydroxy-3-methylbut-2-enyl diphosphate
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references [25] and [26]. the pathway.
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terminal step of the 2-C-methyl-D-erythritol-4-phosphate pathway
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diffraction studies of recombinant Escherichia coli 4-
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S, Bacher A, Eisenreich W, Rohdich F, Hunter WN: Biosynthesis of
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erythritol kinase. Proc Natl Acad Sci USA (2003) 100(16):9173-9178.
47. Wolff M, Seemann M, Tse Sum Bui B, Frapart Y, Tritsch D, Estrabot
AG, Rodriguez-Concepcin M, Boronat A, Marquet A, Rohmer M:
62. Wada T, Kuzuyama T, Satoh S, Kuramitsu S, Yokoyama S, Unzai S,
Isoprenoid biosynthesis via the methylerythritol phosphate
Tame JR, Park SY: Crystal structure of 4-(cytidine 5'-diphospho)-2-
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Update on Biochemistry of Plant Volatiles
Plants have a penchant for perfuming the atmo- Major progress in plant volatile research, as in other
sphere around them. Since antiquity it has been areas of plant biology, has come from the use of
known that both floral and vegetative parts of many molecular and biochemical techniques. A large number
species emit substances with distinctive smells. The of genes encoding enzymes of volatile biosynthesis
discovery of the gaseous hormone ethylene 70 years have recently been reported. In vitro characterization of
ago brought the realization that at least some of the the heterologously expressed enzymes, especially de-
compounds emitted may have physiological signifi- termination of their substrate and product specificity,
cance without any distinctive smell to humans. At has helped clarify the pathways of volatile formation.
present, more than 1,000 low Mr organic compounds In addition, investigation of the spatial and temporal
have been reported to be emitted from plants, al- patterns of gene expression has provided new infor-
though a comprehensive list is available only for floral mation on the factors regulating the emission of plant
volatiles (Knudsen et al., 1993). volatile compounds. In this update, we survey the
Our knowledge of the occurrence and distribution of latest advances on the biosynthesis and regulation of
plant volatiles has been significantly extended in the plant volatiles, beginning with a brief review of the
last 15 years thanks to the adoption of simple, sensitive function of these substances.
methods for headspace sampling and the availability of
relatively inexpensive bench-top instruments for gas
chromatography-mass spectrometry. The substances FUNCTION OF PLANT VOLATILES
reported are largely lipophilic products with molecular
masses under 300. Most can be assigned to the follow- Perhaps the greatest mysteries surrounding vola-
ing classes (in order of decreasing size): terpenoids, tiles concern their function in the life of the plant.
fatty acid derivatives including lipoxygenase path- While it is generally assumed that compounds emitted
way products, benzenoids and phenylpropanoids, from flowers serve to attract and guide pollinators
C5-branched compounds, and various nitrogen and (Reinhard et al., 2004), only scattered attempts have
sulfur containing compounds. Nearly all of these classes been made to demonstrate the ability of individual
are emitted from vegetative parts as well as flowers substances to attract specific pollinators. Many floral
(Knudsen et al., 1993), and some are even emitted from volatiles have anti-microbial or anti-herbivore activity
roots (Steeghs et al., 2004). A major discovery of the last (DeMoraes et al., 2001; Friedman et al., 2002; Hammer
decade is that plants commonly emit much greater et al., 2003), and so could also act to protect valuable
amounts and varieties of volatiles after herbivore damage, reproductive parts of plants from enemies.
and not just from the site of injury (Pare and Tumlinson, Among vegetative volatiles, the most intensively
1999). studied substance is isoprene, a simple five-carbon
terpene emitted from the foliage of many woody spe-
cies (Sharkey and Yeh, 2001b). The function of isoprene
is still controversial, and this compound may act to
1
This work was supported by the U.S. National Science Founda- increase the tolerance of photosynthesis to high tem-
tion (grant nos. MCB0212802 to N.D., MCB0312466 and IBN peratures by stabilizing the thylakoid membranes
0211697 to E.P.), by the U.S. Department of Agriculture (grant no. (Sharkey et al., 2001a) or by quenching reactive oxygen
20033531813619 to N.D.), by the U.S. Israel Binational Agriculture species (Loreto and Velikova, 2001). The release of
Research and Development funds (grant nos. US343703 to N.D. volatiles from vegetative organs following herbivore
and IS333202 to E.P.), by the Fred Gloeckner Foundation, Inc. (to damage seems to be a general property of plant spe-
N.D.), by the German National Science Foundation (DE837/21 to cies. Contributions to this special issue cover herbivore-
J.G.), by the European Commission (QLK3CT200201930 and
induced volatiles from cabbage (Brassica oleracea;
MRTNCT2003504720 to J.G.), and by the Max Planck Society (to
J.G.). This paper is contribution Number 17438 from Purdue Uni- Vuorinen et al., 2004b), cucumber (Cucumis sativus;
versity Agricultural Experimental Station. Mercke et al., 2004), Lotus japonicus (Arimura et al.,
* Corresponding author; e-mail [email protected]; fax 2004b), and maize (Zea mays; Degen et al., 2004). These
17654940391. substances have been demonstrated to serve as indirect
www.plantphysiol.org/cgi/doi/10.1104/pp.104.049981. plant defenses. That is, they attract arthropods that
Plant Physiology, August 2004, Vol. 135, pp. 18931902, www.plantphysiol.org 2004 American Society of Plant Biologists 1893
Dudareva et al.
prey upon or parasitize herbivores, thus minimizing 2003), particularly in the direction from plastids to
further damage to plant tissue (Pare and Tumlinson, cytosol (Laule et al., 2003). This issue contains two
1999; Dicke and Van Loon, 2000). In some cases, contributions concerning the regulation of the basic
herbivore-induced volatiles may also act as direct pathways in relation to isoprene formation. Although
defenses, repelling (DeMoraes et al., 2001; Kessler and produced largely by the plastidial pathway, isoprene
Baldwin, 2001) or intoxicating (Vancanneyt et al., 2001) also seems to arise from extra-plastidial sources, but
herbivores and pathogens (Andersen et al., 1994). The there is apparently no cross-talk between the two
possibility that these substances also act in plant-plant pathways in its formation (Loreto et al., 2004a). The
communication has been discussed (Arimura et al., plastidial pathway is controlled by tight feedback
2000; Dicke and Bruin, 2001; Engelberth et al., 2004). regulation on its first step, deoxyxylulose-5-phosphate
Herbivore-induced volatiles could additionally synthase (Wolfertz et al., 2004).
have physiological roles within the plant, with their In the second phase of terpene biosynthesis, IPP and
release being a consequence of their volatility and DMAPP condense to form geranyl diphosphate (GPP),
membrane solubility. Like isoprene, some herbivore- farnesyl diphosphate (FPP), and geranylgeranyl di-
induced monoterpenes and sesquiterpenes have the phosphate, the precursors of monoterpenes, sesquiter-
potential to combine with various reactive oxygen penes, and diterpenes, respectively. These reactions are
species (Hoffmann et al., 1997; Bonn and Moortgat, catalyzed by short-chain prenyltransferases (Koyama
2003), and so could protect against internal oxidative and Ogura, 1999; Liang et al., 2002). FPP is synthesized
damage (Delfine et al., 2000; Loreto et al., 2004b). In by a large family of homodimeric prenyltransferases
fact, ozone fumigation has recently been reported to called FPP synthases. However, the situation regarding
promote the emission of herbivore-induced volatiles GPP formation is more complex. While the GPP syn-
(Vuorinen et al., 2004a). Yet, it is still unclear why thases of Arabidopsis (Bouvier et al., 2000) and grand
oxidative stress is likely to be significantly higher after fir (Abies grandis; Burke and Croteau, 2002) are homo-
herbivore damage. Further studies are needed to help dimers, like other short-chain prenyltransferases, those
elucidate the roles of these and other plant volatiles. reported from peppermint (Mentha 3 piperita) leaves
The growing number of reports on genes involved in (Burke et al., 1999) and the flowers of snapdragon
volatile formation, as described in the following sec- (Antirrhinum majus) and Clarkia breweri (Tholl et al.,
tions, should enable investigators to manipulate vola- 2004) are unusual heterodimeric enzymes, with each
tile emission and test its function in plants. subunit being a member of the prenyltransferase pro-
tein family.
The third phase of terpene volatile biosynthesis
involves the conversion of the various prenyl diphos-
BIOSYNTHESIS OF VOLATILE TERPENES phates, DMAPP (C5), GPP (C10), FPP (C15), and gera-
nylgeranyl diphosphate (C20), to hemiterpenes
Terpenes, as the largest class of plant secondary (isoprene and 2-methyl-3-buten-2-ol), monoterpenes,
metabolites, have many volatile representatives. The sesquiterpenes, and diterpenes, respectively. These
majority of hemiterpenes (C5), monoterpenes (C10), reactions, carried out by a large family of enzymes
sesquiterpenes (C15), and even some diterpenes (C20) known as terpene synthases (Cane, 1999; Wise and
have high enough vapor pressures at normal atmo- Croteau, 1999), produce the primary representatives of
spheric conditions to allow significant release into the each skeletal type. The investigation of terpene syn-
air. The basic pathway of volatile terpenoid biosynthe- thases is a very active area of plant volatile research and
sis is conveniently treated in three phases: (1) formation this issue contains four contributions describing the
of the basic C5 units, (2) condensation of two or three C5 isolation of genes of this type from Norway spruce
units to form C10, C15, or C20 prenyl diphosphates, and (Picea abies; Martin et al., 2004), Arabidopsis (Chen et al.,
(3) conversion of the resulting prenyl diphosphates to 2004), cucumber (Mercke et al., 2004), and L. japonicus
end products. (Arimura et al., 2004b). These gene sequences give new
The formation of basic C5 units, isopentenyl diphos- insights into the evolutionary origin and genetic regu-
phate (IPP) and dimethylallyl diphosphate (DMAPP) lation of terpene synthases. One of the most outstand-
proceeds via two alternative pathways: the long known ing properties of these enzymes is their proclivity for
mevalonate pathway from acetyl-CoA and the meth- making multiple products from a single substrate.
ylerythritol phosphate pathway from pyruvate and Hence, there has been much curiosity about the carbo-
glyceraldehyde-3-phosphate, discovered only in the cationic reaction mechanism. The elucidation of the
last 10 years (for review, see Rodriguez-Concepcion first crystal structures of plant terpene synthases
and Boronat, 2002). The methylerythritol phosphate (Starks et al., 1997; Whittington et al., 2002) now puts
pathway, localized in the plastids, is thought to provide this work on a much stronger experimental footing.
IPP and DMAPP for hemiterpene, monoterpene, and Many terpene volatiles are direct products of terpene
diterpene biosynthesis, while the cytosol-localized me- synthases, but others are formed through transforma-
valonate pathway provides C5 units for sesquiterpene tion of the initial products by oxidation, dehydrogena-
biosynthesis. However, metabolic cross-talk be- tion, acylation, and other reaction types. These are
tween the two pathways is prevalent (Schuhr et al., discussed in the following section.
1894 Plant Physiol. Vol. 135, 2004
Update on Biochemistry of Plant Volatiles
leaf volatiles, the mixtures of compounds that are 3,5-Dimethoxytoluene, a major scent compound in
emitted when the leaf is damaged (Pichersky and many hybrid roses, is produced from orcinol (3,5-
Gershenzon, 2002). In addition to the C6 aldehyde, dihydroxytoluene) in two successive methylation re-
cleavage of linoleic acid at the 12 to 13 bond also actions catalyzed by two very similar MTs, orcinol
produces a C12 compound that can subsequently be OMTs (OOMT1 and OOMT2; Lavid et al., 2002; Scalliet
converted to jasmonic acid (Howe and Schilmiller, et al., 2002). Both enzymes can carry out both reac-
2002). While jasmonic acid is not by itself volatile, its tions; however, OOMT1 is more catalytically efficient
methylester is (see below). with orcinol while OOMT2 is more catalytically effi-
cient with 3-methoxy,5-hydroxytoluene (Lavid et al.
Oxidation by Dehydrogenases 2002). Chinese rose (Rosa chinensis) flowers make
a similar compound with three methoxyl groups,
NADP/NAD-dependent oxidoreductases are an- 1,3,5-trimethoxybenzene, which is synthesized from
other large and well-studied family of proteins with 1,3,5-trihydroxybenzene. OOMT1 and OOMT2 can
representatives found to be involved in the biosynthe- catalyze the methylation of the second and third
sis of volatiles. Such enzymes have been implicated in intermediates (1-methoxy,3,5-dihydroxybenzene and
the interconversion of volatile alcohols and aldehydes 1,3-dimethoxy,5-hydroxybenzene) but not the methyl-
(Fig. 1B). For example, apparently nonspecific alcohol ation of 1,3,5-trihydroxybenzene, also known as phlor-
dehydrogenases can convert short-chain aldehydes oglucinol (Lavid et al., 2002; Scalliet et al., 2002). The
such as hexanal and 3-cis-hexenal to hexenol and 3-cis- enzyme that methylates this compound, phlorogluci-
hexenol, alcohols that are also found in damaged nol OMT (POMT), also belongs to the Type I MT
leaves (Bate et al., 1998). This lack of tight substrate family, but is only distantly related to OOMT1 and
specificity was used to alter the aroma profile of ripe OOMT2 (Wu et al., 2004).
tomato (Lycopersicon esculentum) fruit by genetic engi- An important strawberry (Fragaria 3 ananassa)
neering (Prestage et al., 1999). Some terpene alcohols aroma compound, 2,5-dimethyl-4-methoxy-3(2H)-
such as geraniol and carveol are converted to alde- furanone, was shown to be produced by the action of
hydes by similarly nonspecific dehydrogenases (Hal- another Type I MT. This MT appears to be able to
lahan et al., 1995; Bouwmeester et al., 1998). Geranial methylate a wide range of substrates, including inter-
and neral (which are coproduced by the oxidation of mediates of the lignin biosynthetic pathway such as
geraniol; the mixture is termed citral), have lemony coniferal aldehyde and coniferal alcohol (Wein et al.,
aroma and are found in many plants, and carvone 2002).
gives caraway its distinct flavor. And benzyl alcohol, Eugenol, mentioned above as the substrate of a eu-
a major floral scent component in many Nicotianeae genol MT and an important volatile spice on its own,
species (Raguso et al., 2003) and elsewhere, is also and vanillin, another important aroma compound,
likely derived from benzaldehyde in a reversible re- also contain a 3-methoxyl group on their benzene
action catalyzed by a member of the NADP/NAD- ring. The synthetic pathways of these compounds
dependent oxidoreductases family (Fig. 1B; Boatright share the first few steps with the lignin pathway. Gang
et al., 2004). et al. (2002a) demonstrated that eugenol is derived
from a lignin intermediate past the para and meta
Methylation of Hydroxyl Groups hydroxylation reactions and the 3-hydroxyl methyla-
tion, which is catalyzed by CCOMT (caffeoyl-CoA
A large portion of plant volatiles contain a methyl- OMT). This is also likely to be the case for vanillin,
ated hydroxyl group (i.e. a methoxyl group). The although this has not yet been demonstrated conclu-
methyl group is usually added in a reaction catalyzed sively. CCOMT is a member of the Type II MT family of
by a methyltransferase (MT) in which S-adenosyl-L- plants (Noel et al., 2003).
methionnine (SAM) serves as the methyl donor. All
plant methyltransferases appear to share a similar
SAM-binding domain; however, they fall into distinct Methylation of Carboxyl Groups
families that share little primary sequence similarity
elsewhere (Noel et al., 2003). A large family of meth- Some methyl esters are extremely wide spread in the
yltransferases with members involved in the synthesis plant kingdom. For example, methylsalicylate has
of both volatile and nonvolatile small molecules (as been reported in numerous floral scents (Knudsen
opposed to proteins or nucleic acids) has been iden- and Tollsten, 1993), and it is also commonly emitted
tified and designated as the Type I methyltransferase from vegetative tissues under attack by insects or
family (Noel et al., 2003). Members of this MT family parasites (Van Poecke et al., 2001; Chen et al., 2003). An
have been shown to catalyze the 4-hydroxyl methyl- enzyme capable of methylating salicylic acid (SA),
ation of eugenol to form methyleugenol in flowers of salicylic acid carboxyl methyltransferase (SAMT) was
C. breweri and in the glands of basil (Ocimum basilicum), first reported from C. breweri flowers (Fig. 1D; Ross
and also the methylation of chavicol to methylchavicol et al., 1999). It has since been identified from several
in the basil glands (Fig. 1C; Wang et al., 1997; Lew- other plant species (Fukami et al., 2002; Negre et al.,
insohn et al., 2000; Gang et al., 2002b). 2002, 2003; Pott et al., 2002, 2004; Chen et al., 2003).
1896 Plant Physiol. Vol. 135, 2004
Update on Biochemistry of Plant Volatiles
This enzyme, which uses SAM as the methyl donor, The BAHD enzymes often show wide substrate
defines a new type of plant MT known as Type III or specificity for both the acyl moiety and the alcohol
SABATH MT (after the first two letters of the names of moiety. For example, the petunia benzyl alcohol
the first three enzymes identified in this family). Some benzoyl-CoA transferase enzyme can also transfer an
SAMT enzymes have been shown to be able to meth- acetyl moiety to the alcohol phenylethanol, producing
ylate also benzoic acid (BA), a compound identical to phenylethylacetate (Boatright et al., 2004). Similarly,
SA except for lacking the 2-hydroxyl group present in a BAHD enzyme from ripening strawberry (Fragaria
SA (for example, Pott et al., 2004). On the other hand, spp) fruit, can use a series of acyl moieties such as
benzoic acid carboxyl methyltransferase (BAMT), the acetyl, butanyl, and hexanyl, and transfer them to
enzyme responsible for the snapdragon floral volatile various alcohols such as heptanol, octanol, and gera-
methylbenzoate, cannot methylate SA (Dudareva et al., niol (Aharoni et al., 2000; Beekwilder et al., 2004). An
2000; Murfitt et al., 2000). acyltransferase from banana (Musa sapientum) has
There are 24 SABATH MTs encoded by the Arabi- similarly wide substrate specificity (Beekwilder et al.,
dopsis genome, including one enzyme that methylates 2004), and a rose (Rose hybrida) flower BAHD enzyme
both SA and BA (Chen et al., 2003). It is not known if all can acetylate both geraniol and citronellol (Shalit et al.,
of these MTs are involved in the biosynthesis of volatile 2003). In such cases, the type of volatile formed in
compounds, but MTs belonging to the SABATH fam- a given tissue depends more on the internal concen-
ily have been shown to be responsible for the three trations of the substrates than on the Km and Kcat
consecutive metylation reactions in the biosynthesis of values of the enzyme for these substrates (Beekwilder
caffeine, a nonvolatile compound, in Coffea arabica et al., 2004; Boatright et al., 2004).
(Uefuji et al., 2003). However, another Arabidopsis
SABATH MT was shown to methylate jasmonic acid to
The Production of the C6-C1 Benzenoids from
form methyljasmonate (Seo et al., 2001). While this
C6-C3 Phenylpropanoids
molecule may act as an internal signal molecule in
Arabidopsis and other plant species, it is also emitted The shortening by two carbons of the three-carbon
from injured plants (Howe and Schilmiller, 2002), and chain attached to the phenyl ring of phenylpropanoids
has also been reported in the floral scent of several plant leads to the formation of benzenoid compounds. The
species (Knudsen et al., 1993) where it is likely to be mechanism by which this is achieved is not fully
formed by similar enzymes. understood. In vivo stable isotope labeling and com-
puter-assisted metabolic flux analysis, described in
Acylation this issue, revealed that both the CoA-dependent-
b-oxidative and CoA-independent-non-b-oxidative
Acylation, most often with an acetyl moiety but also pathways are involved in the formation of benzenoid
with larger acyls such butanoyl or benzoyl acyls, to compounds in petunia (Boatright et al., 2004). How-
make volatile compounds is also common. In all known ever, a recent discovery also indicates that in the case
examples, such plant volatile esters are synthesized by of 2-hydroxybenzoic acid (i.e. salicylic acid), a third
a recently discovered family of plant acyltransferases pathway, via the isochorismate pathway, may also
called BAHD, after the first letter of the first four operate in plants, as it does in bacteria (Wildermuth
enzymes identified (St-Pierre and De Luca, 2000). The et al., 2001).
basic reaction catalyzed by these enzymes is the trans-
fer of an acyl group from an acyl-CoA intermediate to
the hydroxyl group of an alcohol (Fig. 1E). Many BAHD REGULATION OF EMISSION OF
enzymes are involved in the synthesis of nonvolatile VOLATILE COMPOUNDS
compounds such as acylated alkaloids or taxol deriv-
atives (St-Pierre et al., 1998; Walker and Croteau, 2000), Emission of a particular volatile compound into the
or in early steps of pathways that may lead to the atmosphere depends on both the rate of its biosynthe-
synthesis of volatiles such as eugenol (Gang et al., sis and the rate of its release. Substantial progress in
2002a). BAHD acyltransferases directly involved in the last decade in the isolation and characterization of
volatile synthesis include benzyl alcohol acetyl-CoA genes responsible for the formation of volatile com-
transferase from C. breweri flowers, which produced pounds has facilitated the investigation of the regula-
benzyl acetate (Fig. 1E; Dudareva et al., 1998); benzyl tion of the biosynthesis of plant volatiles. It has been
alcohol benzoyl-CoA transferase, which produces ben- found that volatiles are synthesized de novo in the
zylbenzoate in flowers of Clarkia (DAuria et al., 2002) tissues from which they are emitted. Biosynthesis
and petunia (Petunia hybrida; Boatright et al., 2004), and normally occurs in the epidermal cells of plant tissues
in tobacco mosaic virus-infected leaves of tobacco from which they can escape into the atmosphere or
(Nicotiana tabacum; DAuria et al., 2002); and 3-cis- rhizosphere after being synthesized (Dudareva et al.,
hexen-1-ol acetyl CoA transferase, which produces 1996; Dudareva and Pichersky, 2000; Kolosova et al.,
3-cis-hexenyl acetate (a green leaf volatile) and is in- 2001b; Chen et al., 2004) or in the secretory structures
duced in damaged leaves of Arabidopsis (DAuria et al., or glandular trichomes as, for instance, was found in
2002). peppermint, Artemisia annua, and sweet basil (Ocimum
Plant Physiol. Vol. 135, 2004 1897
Dudareva et al.
basilicum; McCaskill et al., 1992; Gang et al., 2001; Lu engineering. When the linalool synthase gene was
et al., 2002). introduced under the control of the cauliflower mosaic
Formation of volatile compounds is spatially regu- virus 35S constitutive promoter into petunia W115, the
lated. Of the plant organs in scented species, flow- differences between organs in the amount of the syn-
ers produce the most diverse and the highest amount thesized linalool or its glycoside depended more on
of volatile compounds, which peak when the flowers the availability of the substrate GPP in the tissue
are ready for pollination. Vegetative tissue also re- than on the expression of the linalool synthase gene
leases small quantities of volatile organic compounds, (Lucker et al., 2001). In peppermint the up-regulation of
which could be induced by mechanical damage or by 1-deoxy-D-xylulose-5-phosphate reductase, which cat-
herbivore- or pathogen infection (Loughrin et al., 1994; alyzes the conversion of 1-deoxy-D-xylulose-5-phos-
Pare and Tumlinson, 1997; Arimura et al., 2004a). In phate to methylerythritol phosphate, increased the flux
herbs, such as peppermint, significant amounts of vola- to GPP and led to about a 50% increase in the essential
tile compounds accumulate in the leaf glandular oil production (Mahmoud and Croteau, 2001). Addi-
trichomes and the emitted volatiles represent only a tional regulation of GPP formation can occur at the level
small fraction of the total pool produced (Gershenzon of GPP synthase, as was shown in snapdragon where
et al., 2000). the small subunit can play a key role in GPP biosyn-
Production and emission of volatile compounds is thesis (Tholl et al., 2004). Feedback regulation of GPP
also a developmentally regulated process. Volatile emis- synthase by product and substrate inhibition could also
sion in flowers and accumulation in leaves and fruits contribute to the regulatory control of the flux to GPP
follow similar developmental patterns, increasing dur- and subsequently to monoterpene production (Tholl
ing the early stages of organ development (when leaves et al., 2004). When enzymes competed for the same
are young and not fully expended, fruit is not yet substrate as in the case of three monoterpene synthases
mature, or when flowers are ready for pollination) and (g-terpinene cyclase, [1]-limonene cyclase, and [2]-b-
then either remaining relatively constant or decreasing pinene cyclase) introduced into tobacco plants, the
over the organs lifespan (Bouwmeester et al., 1998; magnitude of monoterpene emission in leaves was
Dudareva and Pichersky, 2000; Gershenzon et al., 2000). close to that predicted based on the Km values of the
The concurrent temporal changes in activities of en- enzymes for GPP, while the emission levels in flowers
zymes responsible for the final steps of volatile forma- were comparable suggesting that the GPP pool did not
tion, enzyme protein levels, and the expression of limit monoterpene production (Lucker et al., 2004).
corresponding structural genes suggest that the devel- These results show that while the investigation of the
opmental biosynthesis of volatiles is regulated largely at regulation of the final steps of volatile biosynthesis is an
the level of gene expression (Dudareva et al., 1996; important starting point, a detailed understanding of
McConkey et al., 2000). It is still unclear to what extent the regulation of the flux through the entire biochemical
transcriptional, posttranscriptional, translational, post- pathway is essential for the complete understanding of
translational, and other events contribute to this process. production and emission of secondary volatile com-
In general, more than one biochemical pathway is pounds.
responsible for a blend of volatile compounds released Emission of volatile compounds from flowers and
from different plant tissues. A comparative analysis of leaves of some plant species, as well as herbivore-
the regulation of benzenoid and monoterpene emis- induced volatiles, varies remarkably throughout the
sion in snapdragon flowers revealed that the orches- photoperiod. The release of floral volatiles in these
trated emission of phenylpropanoid and isoprenoid species displays a rhythmic pattern with maximum
compounds is regulated upstream of individual met- emission during the day or night, which generally
abolic pathways and includes the coordinated expres- coincides with the foraging activities of potential
sion of genes that encode enzymes involved in the pollinators, and is controlled by a circadian clock or
final steps of scent biosynthesis (Dudareva et al., 2000, regulated by light (Jakobsen and Olsen, 1994; Helsper
2003). However, transcription factors that regulate et al., 1998; Kolosova et al., 2001a). Isoprene, as well as
multiple biosynthetic pathways leading to the forma- volatiles emitted from undamaged and herbivore-
tion of odor bouquet have not yet been discovered. attacked leaves, exhibit a distinct diurnal emission
The level of the enzyme responsible for the final step pattern (Loughrin et al., 1994; Loreto et al., 1996; De
of the biosynthesis of a particular volatile is not the only Moraes et al., 2001; Lerdau and Gray, 2003; Martin
limiting factor. The target for the regulation of devel- et al., 2003; Arimura et al., 2004a) with some leaf
opmental production of volatile compounds also in- volatiles emission also controlled by a circadian clock,
cludes the level of supplied substrate in the cell for example (2)-b-pinene in Artemisia annua (Lu et al.,
(Dudareva et al., 2000). In the case of enzymes that are 2002). In flowers the rhythmic production and emis-
able to use several similar substrates, such as SAMTand sion of some volatile compounds, such as the volatile
acyltransferases, the level of supplied precursor con- ester methylbenzoate, is regulated primarily by the
trols the type of produced product (Negre et al., 2003; level of substrate availability (BA), which in turn could
Boatright et al., 2004; Pott et al., 2004). The role of be regulated at the level of expression of genes en-
substrate in the regulation of the biosynthesis of volatile coding the key enzymes of its biosynthesis (Kolosova
compounds was also recently confirmed by metabolic et al., 2001a). While regulation of isoprene emission
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Isoprenoids are a hugely diverse family of compounds derived It is now well established that all the MEP pathway enzymes
from the C5 precursors isopentenyl diphosphate (IPP) and are encoded by the nuclear genome and imported into plas-
dimethylallyl diphosphate (DMAPP). Although all free-living tids. Recent proteomics approaches have detected all the
organisms synthesize isoprenoids, they are particularly abun- Arabidopsis MEP pathway enzymes in the stroma and have
activity of rate-determining enzymes of both pathways are their physiology. In addition to the well-known isoprenoid
controlled by different posttranslational mechanisms involv- hormones (brassinosteroids, cytokinins, gibberellins, and
ing the PP2A phosphatase (for HMGR) and, in the case of MEP abscisic acid), it was recently discovered that strigolactones
pathway enzymes, the stromal ClpPR proteolytic complex (a group of carotenoids-derived metabolites known to
(Flores-Perez etal., 2008, 2010; Leivar etal., 2011; Vranova be exuded from roots) and strigolactone-like compounds
et al., 2012). All these mechanisms (Figure 1) are likely to such as carlactone can also regulate plant development
provide a fine control of isoprenoid biosynthesis, allowing (Gomez-Roldan etal., 2008; Umehara etal., 2008; Alder et al.,
an appropriate supply of the products needed for essential 2012). Most interestingly, other isoprenoid metabolites have
processes such as respiration or photosynthesis but also for been found to modulate plant responses in new, unsuspected
regulatory processes. ways. Recent work has confirmed that methylerythritol
cyclodiphoshate (MEcPP), an intermediate of the MEP
pathway (Figure 1), acts as a signal released from the
REGULATORY ROLES OF ISOPRENOIDS plastids to modulate the expression of stress-related genes
As described above, some isoprenoid metabolites are used (Xiao et al., 2012). Some isoprenoids can regulate protein
by plants as hormones to regulate multiple aspects of function by direct binding. Proteins can be modified by the
966 Pulido et al. New Insights into Plant Isoprenoid Metabolism
Table1. Subcellular localization of the Arabidopsis enzymes involved in the production of isoprenoid precursors. Cyt, cytosol; ER-Cyt,
anchored to the endoplasmic reticulum; Perox, peroxisomes; Plast, plastids; Mit, mitochondria. Data from Arabidopsis Isoprenoid
Pathway Database (www.atipd.ethz.ch/index.php), MASCP Gator Database (http://gator.masc-proteomics.org/), and the indicated
references: 1 Simkin etal., 2011; 2 Joyard etal., 2009; 3 Sapir-Mir etal., 2008.
covalent attachment of C15 (farnesyl diphosphate, FPP) or role as regulators of plant growth and development than
C20 (geranylgeranyl diphosphate, GGPP) isoprenyl groups previously thought.
to C-terminal cysteine residues, in a process known as
protein prenylation. It was well established that prenylation
modulates the interaction of proteins with membranes or
UNDERSTANDING THE COMPLEXITY
other proteins and hence influences their biological role.
OF ISOPRENOID METABOLISM
Moreover, recent results have shown that the accumulation The picture arising from the above described data shows
of farnesylcisteine residues released after degradation of an extremely complex, highly networked system linking
farnesylated proteins can also impact plant responses such isoprenoid metabolism with a variety of other processes.
as abscisic acid sensitivity (Huizinga et al., 2010). Besides Understanding how plants regulate the production of their
prenylation, a large-scale analysis of the proteinmetabolite isoprenoids as a whole (i.e., as a system) remains a major
interactome in yeast reported the binding of different sterols challenge during the coming years. The topology of the
to many proteins, including regulatory kinases (Li etal., 2012). plant isoprenoid pathway network and its dynamics at the
Although the interaction between sterols and plant proteins gene expression level in response to diverse stimuli have
remains to be fully explored, it is becoming clearer that it been recently reviewed (Vranova etal., 2012). However, it is
might have important regulatory consequences. For example, becoming more and more evident that post-transcriptional
sterols appear to facilitate membrane association and hence mechanisms are also essential to precisely control isoprenoid
activity of ARGONAUTE 1, an Arabidopsis protein required metabolism in plant cells (Figure1) (Flores-Perez et al., 2008,
for microRNA function (Brodersen etal., 2012). These results 2010; Huizinga et al., 2010; Paetzold et al., 2010; Leivar et
together suggest that isoprenoids may play a more general al., 2011; Brodersen et al., 2012). Arecent proteomics study
Pulido et al. New Insights into Plant Isoprenoid Metabolism 967
focusing on isoprenoid biosynthesis in plastids has shown Brodersen, P., et al. (2012). Isoprenoid biosynthesis is required
a diverse, sometimes puzzling, distribution of biosynthetic for miRNA function and affects membrane association of
enzymes in a variety of subplastidial compartments, including ARGONAUTE 1 in Arabidopsis. Proc. Natl. Acad. Sci. U S A.
envelopes, thylakoids, stroma, and plastoglobuli (Joyard 109,17781783.
etal., 2009). The wide array of Arabidopsis proteomic data Flores-Perez, F., et al. (2010). PLEIOTROPIC REGULATORY LOCUS
and resources available in public databases should also allow 1 (PRL1) Integrates the Regulation of Sugar Responses with
Isoprenoid Metabolism in Arabidopsis. Mol. Plant. 3,101112.
determination of the localization of all the pathways that
produce isoprenoids in the cytosol, endoplasmic reticulum, Flores-Perez, U., Sauret-Geto, S., Gas, E., Jarvis, P., and
Rodrguez-Concepcin, M. (2008). A Mutant Impaired in
peroxisomes, or mitochondria (Vranova et al., 2012). This
the Production of Plastome-Encoded Proteins Uncovers a
information will help in understanding the connections
Mechanism for the Homeostasis of Isoprenoid Biosynthetic
between the MEP and MVA pathways with the downstream
Enzymes in Arabidopsis Plastids. Plant. Cell. 20,13031315.
routes that lead to the variety of isoprenoids produced in
Gomez-Roldan, V., etal. (2008). Strigolactone inhibition of shoot
plant cells, including those that have a regulatory role
branching. Nature. 455,189194.
(Figure 1). But understanding these dynamic connections
Huizinga, D.H., etal. (2010). Farnesylcysteine lyase is involved in