Elsevier

Phytochemistry

Volume 52, Issue 5, November 1999, Pages 843-854
Phytochemistry

Amorpha-4,11-diene synthase catalyses the first probable step in artemisinin biosynthesis

https://doi.org/10.1016/S0031-9422(99)00206-XGet rights and content

Abstract

The endoperoxide sesquiterpene lactone artemisinin and its derivatives are a promising new group of drugs against malaria. Artemisinin is a constituent of the annual herb Artemisia annua L. So far only the later steps in artemisinin biosynthesis — from artemisinic acid — have been elucidated and the expected olefinic sesquiterpene intermediate has never been demonstrated. In pentane extracts of A. annua leaves we detected a sesquiterpene with the mass spectrum of amorpha-4,11-diene. Synthesis of amorpha-4,11-diene from artemisinic acid confirmed the identity. In addition we identified several sesquiterpene synthases of which one of the major activities catalysed the formation of amorpha-4,11-diene from farnesyl diphosphate. This enzyme was partially purified and shows the typical characteristics of sesquiterpene synthases, such as a broad pH optimum around 6.5–7.0, a molecular mass of 56 kDa, and a Km of 0.6 μM. The structure and configuration of amorpha-4,11-diene, its low content in A. annua and the high activity of amorpha-4,11-diene synthase all support that amorpha-4,11-diene is the likely olefinic sesquiterpene intermediate in the biosynthesis of artemisinin.

Introduction

Malaria is an infectious disease caused by protozoa of the genus Plasmodium, which are carried by mosquitoes of the genus Anopheles. The most severe form of malaria, Malaria tropica, is responsible for 300–500 million clinical cases each year of which about 90% occur in Africa. It is estimated that malaria causes between 1.5 and 3 million deaths per year, mainly African children (Butler, 1997). In the past, the fight against malaria was based on two strategies: (i) The extermination of the mosquito vector with pesticides such as DDT. This approach was stopped because of the emergence of mosquito resistance against DDT and the undesirable side effects such as pollution of rivers. As a consequence the occurrence of malaria is now spreading. (ii) The large scale use of quinine and chloroquine for the treatment of patients with malaria and as prophylaxe. This has evoked resistance of the protozoa against these drugs making the fight against malaria more difficult, and malaria now forms an increasing problem in the (sub)tropics. Moreover, it is anticipated that due to global warming, malaria will spread to countries, such as Southern Europe and the USA. Therefore, there is a continuous search for new remedies against malaria.

A very promising new group of drugs against malaria are the endoperoxide sesquiterpene lactone artemisinin and its derivatives (Fig. 1). Trials with artemisinin and derivatives were all very promising, and artemisinin derivatives are now being marketed (Van Geldre, Vergauwe, & Van den Eeckhout, 1997). Artemisinin, also known as qinghaosu, is a constituent of the traditional Chinese medicinal herb Artemisia annua L. (Asteraceae). The earliest mention of this herb dates back to 168 BC in the ancient recipes found in the tomb of the Mawangdui Han dynasty (Klayman, 1985). Its antimalarial activity was described as early as 1596 by Li Shizhen in his Ben Cao Gang Mu (Compendium of Materia Medica) (Klayman, 1985). The active principle was isolated and identified as artemisinin in 1972. Since then there has been a tremendous scientific and commercial interest in this rediscovered antimalarial compound.

Artemisinin is a structurally complex compound and so far the plant A. annua is the only commercially feasible source of artemisinin for drug formulations (Van Geldre et al., 1997). A. annua is a cosmopolitan species, growing wild in many countries, e.g. in China and Vietnam, the Balkan, the former Soviet Union, Argentina and Southern Europe (Van Geldre et al., 1997), and large differences exist in artemisinin content between different strains of A. annua (Delabays et al., 1993, Woerdenbag et al., 1993). A substantial increase in the content of artemisinin would be required to make artemisinin available on a large scale also to the people in The Third World. Selection for high producing lines and traditional breeding, and research on the effects of environmental conditions and cultural practices could perhaps lead to an improvement of artemisinin content (Delabays et al., 1993, Ferreira et al., 1995, Gupta et al., 1996, Laughlin, 1994). A biotechnological approach could be another way to increase artemisinin content in A. annua. However, the biosynthetic pathway of artemisinin has not been completely resolved. Several authors have demonstrated that A. annua converts artemisinic (also named arteannuic) acid and dihydroartemisinic (also named dihydroarteannuic) acid to artemisinin (Kim & Kim, 1993, Sangwan et al., 1993a, Wallaart et al., 1999) (Fig. 1). Akhila et al., 1987, Akhila et al., 1990 hypothesised a pathway in which the formation from farnesyl diphosphate of an unidentified enzyme-bound sesquiterpene-like intermediate represents the first committed step in the biosynthesis of artemisinin.

In addition, many authors have analysed extracts of A. annua to search for possible intermediates in the biosynthesis of artemisinin. Artemisinic and dihydroartemisinic acid were reported by many authors, as well as many olefinic mono- and sesquiterpenes and putative intermediates en route from dihydroartemisinic acid to artemisinin (Ahmad & Misra, 1994, Brown, 1994, Charles et al., 1991, Jung et al., 1986, Jung et al., 1990, Ranasinghe et al., 1993, Woerdenbag et al., 1993; Wallaart, T. E., Pras, N. & Quax, W. J., personal communication). However, none of the reported olefinic sesquiterpenes seemed to fit in the biosynthetic pathway, nor was a possible intermediate between the sesquiterpene olefin and artemisinic acid ever detected, with the exception of artemisinic alcohol which was tentatively identified in the roots of A. annua by Woerdenbag et al. (1993).

It has been postulated that the cyclisation of the ubiquitous precursors geranyl diphosphate, farnesyl diphosphate (FDP) and geranylgeranyl diphosphate to the respective olefinic mono-, sesqui- and diterpene skeletons represents the regulatory step in the biosynthesis of terpenoids (Gershenzon & Croteau, 1990, McCarvey & Croteau, 1995). The accumulation of artemisinic and dihydroartemisinic acid and the absence of any intermediates en route from FDP to these two compounds support that the first step(s) in the biosynthetic pathway of artemisinin (and again some step(s) from (dihydro)artemisinic acid to artemisinin) are indeed regulatory/rate-limiting. In the present work we studied the identity of the probable olefinic sesquiterpene intermediate in the biosynthesis of artemisinin, and isolated and characterised the sesquiterpene synthase that catalyses its formation from FDP (Fig. 1).

Section snippets

Identification of olefinic sesquiterpenes in A. annua leaves

Several authors have analysed hydrocarbon sesquiterpenes in A. annua tissues (Ahmad & Misra, 1994, Charles et al., 1991, Woerdenbag et al., 1993), but a compound that could be the olefinic sesquiterpene intermediate in the biosynthesis of artemisinin was never identified. Fig. 2 shows the results of our analyses of leaf extracts of greenhouse-grown A. annua on two different GC-columns. Results with field-grown plants were similar (data not shown). Of the 14 sesquiterpene olefins that were

Discussion

For the first time the presence of amorpha-4,11-diene (1) in an A. annua solvent extract was demonstrated. The structure and configuration of this compound make it the likely olefinic sesquiterpene intermediate in the biosynthesis of artemisinin (Fig. 1). Moreover, the enzyme activity that catalyses the formation of 1 from FDP is the major sesquiterpene synthase activity in A. annua leaf extracts. The enzyme catalysing the formation of 1 from FDP has the typical characteristics of sesquiterpene

Plant material

Seeds of Artemisia annua L. (Asteraceae) of Vietnamese origin were obtained from Artecef BV (Maarssen, The Netherlands). Taxonomically verified specimens are deposited at the Department of Pharmaceutical Biology, Groningen University, The Netherlands and at the Institute of Materia Medica, Hanoi, Vietnam. Plants were grown on an experimental field in Buitenpost, The Netherlands. A second batch of plants was grown in a greenhouse in 5-l plastic pots containing potting compost at 21/18°C (16/8

Acknowledgements

The authors wish to thank Ch. B. Lugt for his gift of A. annua seeds, Adrie Kooijman for raising the plants, and Jacques Davies and Francel Verstappen for technical assistance.

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