Studies on the pyrogenic decomposition of rubber led workers in the latter half of the nineteenth century to believe that isoprene could be regarded as a fundamental building block for this material. As a result
BASIC METABOLIC PATHWAYS AND THE ORIGIN OF SECONDARY METABOLITES 163
18
Limonene Camphor O
Monoterpenes
Rubber β-Carotene
Carotenoids
Isoprene C CH
CH2 H3C
n
2
H2C
Triterpenes COOH
O
HO
Glycyrrhetinic acid
Sesquiterpenes Steroids Squalene
Zingiberene Fig. 18.17
Application of the isoprene rule illustrating incorporation of C5 units.
of the extensive pioneering investigations into plant terpene structures, Ruzicka published in 1953 his ‘biogenetic isoprene rule’, which indi- cated that the apposition of isoprenoid units could be used to explain not only the formation of rubber and the monoterpenes, but also many other natural products, including some, such as sterols and triterpenes, with complex constitutions. The value of the rule lay in its broad unify- ing concept, which allowed the postulation of a rational sequence of events which might occur in the biogenesis of these otherwise unre- lated compounds. Examples of various structures to which the rule can be applied are indicated in Fig. 18.17.
The task set biochemists was to investigate the validity of the rule, and the work on this subject constitutes a brilliant example of modern biochemistry; however, as will be seen below, this chapter of research is still unfinished. By 1951 it had been established that acetic acid was intimately involved in the synthesis of cholesterol, squalene, yeast sterols and rubber. The use of methyl- and carboxyl-labelled acetic acid with animal tissues indicated that the methyl and carboxyl car- bons alternated in the skeleton of cholesterol or squalene and that the lateral carbon atoms all arose from the methyl group of acetic acid.
The discovery, in 1950, of acetyl-coenzyme A, the so-called ‘active acetate’, gave further support to the role of acetate in biosynthetic processes.
The mevalonic acid pathway. The next major advance in the eluci- dation of the isoprenoid biosynthetic route was the discovery in 1956 of mevalonic acid and the demonstration of its incorporation, by liv- ing tissues, into those compounds to which the isoprene rule applied.
Mevalonic acid (3,5-dihydroxy-3-methylvaleric acid) is a C6 acid and, as such, is not the ‘active isoprene’ unit which forms the basic build- ing block of the isoprenoid compounds. During the next four years, by research involving the use of tracer techniques, inhibitor studies, cell-free extracts, partition chromatography and ionophoresis as well as synthetic organic chemistry, it was established that the C5 compound for which biochemists had been seeking so long was isopentenyl pyrophosphate;
it is derived from mevalonic acid pyrophosphate by decarboxylation and dehydration. Isoprenoid synthesis then proceeds by the condensation of isopentenyl pyrophosphate with the isomeric dimethylallyl pyrophos- phate to yield geranyl pyrophosphate. Further C5 units are added by the
18
addition of more isopentenyl pyrophosphate. These preliminary stages in the biosynthesis of isoprenoid compounds are shown in Fig. 18.18.
From geranyl and farnesyl pyrophosphates various structures can be built up (see Fig. 18.19).
Studies, particularly by Cornforth and Popják, involving the use of stereospecifically 3H- and 14C-labelled mevalonic acid, have dem- onstrated the stereochemical mechanism of the initial stages of iso- prenoid formation. Only the (R)-form of mevalonic acid gives rise to the terpenoids, the (S)-form appearing to be metabolically inactive. In the formation of isopentenyl pyrophosphate, the elimination is trans and the elimination of the proton in the isomerization to the dimethy- lallyl pyrophosphate is also stereospecific (Fig. 18.20A).
In the subsequent additions of the C5 isopentenyl pyrophosphate units to form the terpenoids the elimination of hydrogen is trans.
Figure 18.20B shows the stereochemistry of the addition of one iso- pentenyl pyrophosphate unit. In the biogenesis of rubber, however, the hydrogen elimination produces a cis double bond (Fig. 18.20C).
It is considered that a simple change in orientation of the isopentenyl pyrophosphate on the enzyme surface could produce this change with- out altering the reaction mechanism. In neither rubber nor gutta are hybrid molecules containing both types of bond detectable. The first
direct evidence for the presence of isopentenyl diphosphate isomerase in rubber latex was reported in 1996 (T. Koyama et al., Phytochemistry, 1996, 43, 769).
In recent years the enzymology of the isoprenoid pathway has been extensively studied and for details the reader is referred to a standard text on plant biochemistry. One key regulatory enzyme is hydroxy- methylglutaryl-CoA reductase (EC 1.1.1.34, mevalonate kinase); it has been extensively studied in animals and more recently in plants. As with many enzymes the situation is complicated by the existence of more than one species of enzyme and a plant may possess multiple forms each having a separate subcellular location associated with the biosynthesis of different classes of terpenoids. For a review of the functions and properties of the important isomerase enzyme isopente- nyl diphosphate isomerase see ‘Further reading’.
It should be noted that some metabolites of mixed biogenetic origin involve the mevalonic acid pathway; prenylation for example is com- mon, with C5, C10 and C15 units associated with flavonoids, coumarins, benzoquinones, cannabinoids, alkaloids, etc.
The validity of the mevalonate pathway in the formation of all the major groups noted in Fig. 18.19 has been shown, and until recently no other biosynthetic route to isoprenoids had been discovered.
−OOC CH2 CH2 CH3
CH2
CH2 CH2
CH
CH CH CH
CH2 C CH2 CH3
−CO2
CH3
CH3
CH3 CH3
CH2 CH2
CH3
CH3
CH3 CH3 CH3
CH2 CH2 CH2 CH2 CH3
CH2COO− ATP
COSCoA 2NADPH
Mevalonate
5-Phosphomevalonate
Geranyl pyrophosphate + Isopentenyl pyrophosphate 5-Pyrophosphomevalonate
Isopentenyl pyrophosphate
Dimethylallyl pyrophosphate
Farnesyl pyrophosphate ATP
OH ATP
O3POCH2
CH3
COO−
OH C
C
C
C CH C CH CH2OP2O6−
CH2OP2O6- - - C
C C C
OH Acetyl-CoA
CH3COCH2COSCoA Acetoacetyl-CoA
O6P2OCH2
O6P2OCH2
CH2OP2O6- - -
Hydroxymethylglutarate +
CH3 COSCoA
HOCH2 CH2 CH2 CH3
C COO−
OH
Fig. 18.18
Preliminary stages in the biosynthesis of isoprenoid compounds.
BASIC METABOLIC PATHWAYS AND THE ORIGIN OF SECONDARY METABOLITES 165
18
The 1-deoxy-d-xylulose (triose/pyruvate) pathway. Following its discovery in 1956 mevalonic acid came to be considered the essential precursor for all isoprenoid syntheses. However, in 1993 M. Rohmer et al., (Biochem. J., 1993, 295, 517) showed that a non- mevalonate pathway existed for the formation of hopane-type triter- penoids in bacteria. The novel putative precursor was identified as 1-deoxy-d-xylulose-5-phosphate, formed from glucose via condensation of pyruvate and glyceraldehyde-3-phosphate. Subsequent steps includ- ing a skeletal rearrangement afford isopentenyl pyrophosphate—the same methyl-branched isoprenoid building block as formed by the MVA route.
It was soon demonstrated that this novel route to IPP was also opera- tive in the formation of monoterpenes (Mentha piperita, Thymus vul- garis), diterpenes (Ginkgo biloba, Taxus chinensis) and carotenoids (Daucus carota). This raised the question of to what extent the two pathways co-existed in the plant and it was hypothesized that the classi- cal acetate/mevalonate pathway was a feature of cytoplasmic reactions whereas the triose/pyruvate sequence was a characteristic of the plas- tids. This did not exclude either the movement of plastid-synthesized IPP and DMAPP from the organelle to the cytoplasm or the transloca- tion of a suitable C5-acceptor to the plastid. Evidence accumulating indicates a cooperative involvement of both pathways. Indeed recent work on the biosynthesis of the isoprene units of chamomile sesquit- erpenes (K.-P. Adam and J. Zapp, Phytochemistry, 1998, 48, 953) has shown that for the three C5 units of both bisaboloxide A and chamazu- lene, two were mainly formed by the non-mevalonate pathway and the third was of mixed origin.
The deoxyxylulose (DOX) pathway has helped explain the previ- ously reported rather poor incorporations of MVA into certain iso- prenoids. Thus V. Stanjek et al. (Phytochemistry, 1999, 50, 1141) have obtained a good incorporation of labelled deoxy-d-xylulose into the prenylated segment of furanocoumarins of Apium graveolens leaves, suggesting this to be the preferred intermediate.
Fig. 18.21 illustrates how [1-13C]-glucose, when fed to plants, can be used to differentiate IPP and subsequent metabolites, formed either by the MVA pathway or the DOX route.