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. 2001 Apr;125(4):1930-40.
doi: 10.1104/pp.125.4.1930.

Biosynthesis of germacrene A carboxylic acid in chicory roots. Demonstration of a cytochrome P450 (+)-germacrene a hydroxylase and NADP+-dependent sesquiterpenoid dehydrogenase(s) involved in sesquiterpene lactone biosynthesis

J W de Kraker  1 , M C Franssen, M C Dalm, A de Groot, H J Bouwmeester
Affiliations

Biosynthesis of germacrene A carboxylic acid in chicory roots. Demonstration of a cytochrome P450 (+)-germacrene a hydroxylase and NADP+-dependent sesquiterpenoid dehydrogenase(s) involved in sesquiterpene lactone biosynthesis

J W de Kraker et al. Plant Physiol. 2001 Apr.

Abstract

Sprouts of chicory (Cichorium intybus), a vegetable grown in the dark, have a slightly bitter taste associated with the presence of guaianolides, eudesmanolides, and germacranolides. The committed step in the biosynthesis of these compounds is catalyzed by a (+)-germacrene A synthase. Formation of the lactone ring is the postulated next step in biosynthesis of the germacrene-derived sesquiterpene lactones. The present study confirms this hypothesis by isolation of enzyme activities from chicory roots that introduce a carboxylic acid function in the germacrene A isopropenyl side chain, which is necessary for lactone ring formation. (+)-germacrene A is hydroxylated to germacra-1(10),4,11(13)-trien-12-ol by a cytochrome P450 enzyme, and is subsequently oxidized to germacra-1(10),4,11(13)-trien-12-oic acid by NADP+-dependent dehydrogenase(s). Both oxidized germacrenes were detected as their Cope-rearrangement products elema-1,3,11(13)-trien-12-ol and elema-1,3,11(13)-trien-12-oic acid, respectively. The cyclization products of germacra-1(10),4,11(13)-trien-12-ol, i.e. costol, were also observed. The (+)-germacrene A hydroxylase is inhibited by carbon monoxide (blue-light reversible), has an optimum pH at 8.0, and hydroxylates beta-elemene with a modest degree of enantioselectivity.

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Figures

Figure 1
Figure 1
The major sesquiterpene lactones of chicory: the guaianolides lactucin (1), 8-deoxylactucin (2), and lactupicrin (3); the eudesmanolides sonchuside C (4) and cichoriolide A (5); and the germacranolides sonchuside A (6) and cichorioside C (7).
Figure 2
Figure 2
Proposed biosynthetic route from (+)-germacrene A (8) to (+)-costunolide (12) via germacra-1(10),4,11(13)-trien-12-ol (9), germacra-1(10),4,11(13)-trien-12-al (10), and germacra-1(10),4,11(13)-trien-12-oic acid (11). At the right side of the dotted line, compounds are shown that can be formed from these unstable germacrenes: the heat-induced Cope-rearrangement products (-)-β-elemene (13), (-)-elema-1,3,11(13)-trien-12-ol (14), (-)-elema-1,3,11(13)-trien-12-al (15), elema-1,3,11(13)-trien-12-oic acid (16), and dehydrosaussurea lactone (17); and the acid-induced cyclization products selinene (18) (γ-selinene is usually named selina-4, 11-diene), costol (19), costal (20), and costic acid (21). Compounds with underlined numbers have all been identified in costus roots; (+)-germacrene A (8) and germacra-1(10),4,11(13)-trien-12-al (10) were isolated from other plant species. Note that after hydroxylation the numbering of carbon atoms 12 and 13 is inverted.
Figure 3
Figure 3
Radio-GC analyses of the products formed in incubations of a 20,000g supernatant from chicory roots with [3H]FPP in the absence (A) or presence (B) of an NADPH-regenerating system. In the presence of NADPH the produced [3H]germacrene A (8; germ A) converted into a more polar product. C shows the response of the flame ionization detector (FID) to a standard solution of trans,trans-farnesol (FOL) and (-)-elema-1,3,11(13)-trien-12-ol (14) (EOL), injected under the same GC conditions.
Figure 4
Figure 4
Identification by GC-MS of the products formed by a 20,000g supernatant from chicory roots incubated with (-)-elema-1,3,11(13)-trien-12-ol (14) (EOL) in the absence (A) or presence of NADP+ (B); or with (-)-elema-1,3,11(13)-trien-12-al (15) (EAL) and NAD+ (C). The produced elema-1,3,11(13)-trien-12-oic acid (16) (EAc) has the same retention time as the synthesized standard (D).
Figure 5
Figure 5
A, Radio-GC analyses of the products formed in an incubation of 20,000g supernatant with NADP+ and a pentane-ether extract from the (+)-germacrene A hydroxylase assay of Figure 3B, containing [3H]germacra-1(10),4,11(13)-trien-12-ol (9; GOL) and smaller amounts of [3H]germacrene A (8) (germ A) and [3H]farnesol (FOL). A more polar product is formed together with a minute amount of [3H]farnesal (FAL). Trace B shows the response of the FID to a standard solution of elema-1,3,11(13)-trien-12-oic acid (16) (EAc).
Figure 6
Figure 6
Determination of the stereochemical preference of the (+)-germacrene A hydroxylase using GC-MS equipped with an enantioselective column in selected ion monitoring-mode (m/z 119, 121, 145, 147, and 189). A, Incubation of (±)-β-elemene in the absence of NADPH (blank). B, Incubation of (±)-β-elemene in the presence of NADPH, resulting in a mixture of elema-1,3,11(13)-trien-12-ol (14) enantiomers (EOL). C, Incubation of (-)-β-elemene giving (-)-elema-1,3,11(13)-trien-12-ol. D, Standard of (-)-elema-1,3,11(13)-trien-12-ol (EOL).
Figure 7
Figure 7
Proposed biosynthetical route for the germacrene-derived sesquiterpene lactones present in chicory. I, Cyclization of FPP to (+)-germacrene A (8) by a sesquiterpene synthase. II, Hydroxylation of the isopropenyl side chain by (+)-germacrene A hydroxylase, a cytochrome P450 enzyme. III, Oxidation of germacratrien-12-ol (9) to germacratrien-12-oic acid (11) by NADP+-dependent dehydrogenase(s) via the intermediate germacratrien-12-al (10). IV, The not-yet-demonstrated hydroxylation at the C6-position of germacratrien-12-oic acid and subsequent lactonization to (+)-costunolide (12). V, The postulated formation of guaiane, eudesmane, and germacrane lactones.
Figure 8
Figure 8
Three-dimensional structures of (+)-germacrene A, (-)-β-elemene, and (+)-β-elemene demonstrating the resemblance of these compounds at the site of hydroxylation (C13-position).
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