Euphorbiaceae

Bathe, Ulschan & Tissier, Alain, 2019, Cytochrome P 450 enzymes: A driving force of plant diterpene diversity, Phytochemistry 161, pp. 149-162 : 156

4.3. Gene clusters for diterpene metabolism in Euphorbiaceae are conserved in several species

The Euphorbiaceae family contains 228 plant genera and around 6500 species (www.theplantlist.org). Some family members are used in traditional medicine in Arabia, China, India and in particular in Africa. They are used to treat inter alia epilepsy, anemia, diarrhea, cancer, fungal infections and oedema ( Chhabra et al., 1990). In many cases, bioactivity could be assigned to diterpenoids from these plants ( Beutler et al., 1989). They typically have a macrocyclic structure derived from the casbene, rosane, lathyrane, tigliane or ingenane backbones ( Vasas and Hohmann, 2014). In addition to ethnomedical knowledge, experimental data demonstrated the significance of Euphorbiaceae diterpenoids for therapeutic applications, including against cancer, HIV latent infection or to treat intractable pain ( Corea et al., 2009; King et al., 2014; Lebwohl et al., 2012; Reis et al., 2013). The genes currently known to be involved in Euphorbiaceae diterpene biosynthesis share a high degree of conservation and organization, typically in genetic clusters. For example, Ricinus communis (castor bean) has a biosynthetic cluster with several genes coding for two casbene synthases (CBSs), one neocembrene synthase, six CYP726As (CYP726A13-18), two SDRs, one BAHD acyltransferase, two CYPs from the CYP80C family and one TPSg terpene synthase likely involved in monoterpene biosynthesis. Similar biosynthetic clusters can be found in Euphorbia peplus , revealing genes encoding a CBS and CYP726A19, and on chromosome 1 of Jatropha curcas with three CBSs, five CYP726As (CYP726A20, CYP726A21, CYP726A23, CYP726A24 and CYP726A35), two CYPs from the CYP71D subfamily, four alkenal reductases and three monoterpene synthases ( Boutanaev et al., 2015; King et al., 2014, 2016). Remarkably, these genetic clusters occur in species that belong to distinct major sub-families. It was therefore concluded that the production of Euphorbiaceae diterpenoids evolved early in the emergence of this plant family more than 100 million years ago ( King et al., 2014). During evolution upstream biosynthetic enzymes providing the diterpene backbones (for example casbene, jatrophane or pepluane) kept a high degree of conservation across species because the different backbone types were found in several species ( Shi et al., 2008).

In the pathway of casbene-derived diterpenoids, CYP71 clan subfamily members of CYP726A and CYP71D appear to be the driving force for the production of higher oxidized derivatives ( Fig. 3C ). Some of them are promiscuous and accept not only casbene but also neocembrene. While CYP726A15 from R. communis oxidizes neocembrene at C5, casbene can be oxidized at C5, C6 and C9 by CYP726A14 and CYP726A17-18 from R. communis , by CYP726A20, CYP726A35 and CYP71D495 from J. curcas , by CYP726A27 and CYP71D445 from Euphorbia lathyris , and by CYP726A19, CYP71D365 and CYP726A4 from E. peplus ( Boutanaev et al., 2015; King et al., 2014; Luo et al., 2016). Due to the large number of existing casbene-5-oxidases, this catalytic activity was concluded to be a key step in the biosynthesis of Euphorbiaceae diterpenoids and provides further evidence of a conserved biosynthetic pathway within this plant family ( King et al., 2014). Downstream, an alcohol dehydrogenase from E. lathyris, ADH 1, was recently found to convert 6-keto-5,9-casbenediol into jolkinol C, although the same reaction on the highly similar 6-hydroxy-5,9-diketocasbene can occur spontaneously ( King et al., 2016; Luo et al., 2016). The formation of jolkinol C is characterized by a ring-closure, which is also indispensable for jatrophane, ingenane, myrsinane, daphnane, and lathyrane diterpenes. One can therefore assume that it represents a key step in Euphorbiaceae diterpenoid biosynthesis. However, biosynthetic genes of other Euphorbiaceae diterpenes remain unknown, and further research is necessary to elucidate the pathway of more complex Euphorbiaceae metabolites such as resiniferatoxin, ingenol mebutate and prostratin.

Figures

Fig. 3. CYP activity in diterpene biosynthesis from plants. The substrate positions that are oxi- dized by the given CYPs are highlighted in the sim- plified terpene backbone structures. A, CYP activity in Lamiaceae. CYP76AHs catalyze oxidations of blue and red highlighted positions. X corresponds to a C- atom in rosemary and sage diterpene biosynthesis, and to an O-atom in forskolin biosynthesis from C. forskohlii. B, diterpene CYPs from rice. Some CYPs catalyze oxidations at various positions depending on their substrate. C, Euphorbiaceae CYP activity. The oxidized positions are C5, C6 and C9 in different combinations. D, Gibberellin and steviol bio- syntheses involve activity of CYP714s. E, Diterpene resin acid biosynthesis in conifers include activity of CYP720Bs which oxidize exclusively at position C18. F, CYP725As activity in Taxus sp. The CYP, oxidizing C9, has not been found so far from Taxus sp.