ent -Kaurene is a key intermediate in the biosynthesis of the plant hormone gibberellin. In ent -kaurene biosynthesis in flowering plants, two diterpene cyclases (DTCs), ent -copalyl diphosphate ( ent -CDP) synthase ( ent -CPS) and ent -kaurene synthase (KS), catalyse the cyclization of geranylgeranyl diphosphate to ent -CDP and ent -CDP to ent -kaurene, respectively. In contrast, the moss Physcomitrella patens has a bifunctional ent -CPS/KS (PpCPS/KS) that catalyses both cyclization reactions. To gain more insight into the functional diversity of ent -kaurene biosynthetic enzymes in land plants, we focused on DTCs in the lycophyte Selaginella moellendorffii . The present paper describes the characterization of two S. moellendorffii DTCs (SmKS and SmDTC3) in vitro . SmDTC3 converted ent -CDP into ent -16α-hydroxykaurane and also used other CDP stereoisomers as substrate. Remarkably, SmKS, which produces ent -kaurene from ent -CDP, showed similar substrate selectivity: both SmKS and SmDTC3 synthesized sandaracopimaradiene from normal CDP. Therefore, the diversity of substrate recognition among KSs from other plants was investigated. PpCPS/KS could use normal CDP and syn -CDP as well as ent -CDP as substrate. In contrast, lettuce KS showed high specificity for ent -CDP, and rice KS recognized only ent -CDP. Our studies imply that ancient KS having low substrate specificity has evolved to be specific for ent -CDP to the biosynthesis of gibberellin.

Natural products biosynthesis often requires the action of multiple CYPs (cytochromes P450), whose ability to introduce oxygen, increasing solubility, is critical for imparting biological activity. In previous investigations of rice diterpenoid biosynthesis, we characterized CYPs that catalyse alternative hydroxylation of ent -sandaracopimaradiene, the precursor to the rice oryzalexin antibiotic phytoalexins. In particular, CYP76M5, CYP76M6 and CYP76M8 were all shown to carry out C-7β hydroxylation, whereas CYP701A8 catalyses C-3α hydroxylation, with oxy groups found at both positions in oryzalexins A–D, suggesting that these may act consecutively in oryzalexin biosynthesis. In the present paper, we report that, although CYP701A8 only poorly reacts with 7β-hydroxy- ent -sandaracopimaradiene, CYP76M6 and CYP76M8 readily react with 3α-hydroxy- ent -sandaracopimaradiene. Notably, their activity yields distinct products, resulting from hydroxylation at C-9β by CYP76M6 or C-7β by CYP76M8, on different sides of the core tricyclic ring structure. Thus CYP76M6 and CYP76M8 have distinct non-redundant roles in orzyalexin biosynthesis. Moreover, the resulting 3α,7β- and 3α,9β-diols correspond to oryzalexins D and E respectively. Accordingly, the results of the present study complete the functional identification of the biosynthetic pathway underlying the production of these bioactive phytoalexins. In addition, the altered regiochemistry catalysed by CYP76M6 following C-3α hydroxylation has some implications for its active-site configuration, offering further molecular insight.

The plant-specific phi class of glutathione transferases (GSTFs) are often highly stress-inducible and expressed in a tissue-specific manner, suggestive of them having important protective roles. To date, these functions remain largely unknown, although activities associated with the binding and transport of reactive metabolites have been proposed. Using a sensitive and selective binding screen, we have probed the Arabidopsis thaliana GSTFs for natural product ligands from bacteria and plants. Uniquely, when overexpressed in bacteria, family members GSTF2 and GSTF3 bound a series of heterocyclic compounds, including lumichrome, harmane, norharmane and indole-3-aldehyde. When screened against total metabolite extracts from A. thaliana , GSTF2 also selectively bound the indole-derived phytoalexin camalexin, as well as the flavonol quercetin-3- O -rhamnoside. In each case, isothermal titration calorimetry revealed high-affinity binding (typically K d <1 μM), which was enhanced in the presence of glutathione and by the other heterocyclic ligands. With GSTF2, these secondary ligand associations resulted in an allosteric enhancement in glutathione-conjugating activity. Together with the known stress responsiveness of GSTF2 and its association with membrane vesicles, these results are suggestive of roles in regulating the binding and transport of defence-related compounds in planta .

The evolution of natural product biosynthetic pathways can be envisioned to occur via a number of mechanisms. In the present study we provide evidence that latent plasticity plays a role in such metabolic evolution. In particular, rice ( Oryza sativa ) produces both ent - and syn -CPP (copalyl diphosphate), which are substrates for downstream diterpene synthases. In the present paper we report that several members of this enzymatic family exhibit dual reactivity with some pairing of ent -, syn - or normal CPP stereochemistry. Evident plasticity was observed, as a previously reported ent -sandaracopimaradiene synthase also converts syn -CPP into syn -labda-8(17),12 E ,14-triene, which can be found in planta . Notably, normal CPP is not naturally found in rice. Thus the presence of diterpene synthases that react with this non-native metabolite reveals latent enzymatic/metabolic plasticity, providing biochemical capacity for utilization of such a novel substrate (i.e. normal CPP) which may arise during evolution, the implications of which are discussed.