Involvement of an ent-copalyl diphosphate synthase in tissue-specific accumulation of specialized diterpenes in Andrographis paniculata

Highlights

• Transcripts associated with specialized diterpene accumulation are identified.

• Two class II diterpene synthases with transcriptional variants are characterized.

• ApCPS1 may be involved in general metabolism i.e. gibberellins biosynthesis.

• ApCPS2 is involved in tissue-specific accumulation of specialized diterpenes.

• ApCPS2 is functionally characterized as ent-copalyl diphosphate synthase.

Abstract

Ent-labdane-related diterpene (ent-LRD) specialized (i.e. secondary) metabolites of the medicinal plant kalmegh (Andrographis paniculata) have long been known for several pharmacological activities. However, our understanding of the ent-LRD biosynthetic pathway has remained largely incomplete. Since ent-LRDs accumulate in leaves, we carried out a comparative transcriptional analysis using leaf and root tissues, and identified 389 differentially expressed transcripts, including 223 transcripts that were preferentially expressed in leaf tissue. Analysis of the transcripts revealed various specialized metabolic pathways, including transcripts of the ent-LRD biosynthetic pathway. Two class II diterpene synthases (ApCPS1 and ApCPS2) along with one (ApCPS1′) and two (ApCPS2′ and ApCPS2″) transcriptional variants that were the outcomes of alternative splicing of the precursor mRNA and alternative transcriptional termination, respectively, were identified. ApCPS1 and ApCPS2 encode for 832- and 817-amino acids proteins, respectively, and are phylogenetically related to the dicotyledons ent-copalyl diphosphate synthases (ent-CPSs). The spatio-temporal patterns of ent-LRD metabolites accumulation and gene expression suggested a likely role for ApCPS1 in general (i.e. primary) metabolism, perhaps by providing precursor for the biosynthesis of phytohormone gibberellin (GA). However, ApCPS2 is potentially involved in tissue-specific accumulation of ent-LRD specialized metabolites. Bacterially expressed recombinant ApCPS2 catalyzed the conversion of (E,E,E)-geranylgeranyl diphosphate (GGPP), the general precursor of diterpenes to ent-copalyl diphosphate (ent-CPP), the precursor of ent-LRDs. Taken together, these results advance our understanding of the tissue-specific accumulation of specialized ent-LRDs of medicinal importance.

Introduction

Plants produce a large and diverse array of specialized (i.e. secondary) metabolites that have applications as pharmaceuticals, pesticides, flavours and fragrances [1]. Several specialized metabolites are directly utilized as drugs; however, many are leading models for the development of semisynthetic and synthetic drugs [2]. Specialized metabolites are biosynthesized in plants in tissue-specific, organ-specific, and developmentally-specific ways and also in response to pathogen attack and environmental perturbation; involving highly complex and sophisticated biosynthetic pathways [1]. A comprehensive knowledge of the metabolic pathways and their regulation shall be useful to overcome low product yield of the specialized metabolites in plants, following pathway engineering and molecular breeding approaches [3], [4].

Labdane-related diterpenes (LRDs), with almost 7000 known members comprise a large superfamily of plant compounds with a broad range of biological activities [5], [6]. LRDs possess a characteristic skeleton with a basic decalin structure and an additional C6 skeleton that is either acyclic or constitutes a ring structure with/without an oxygen atom [6], [7]. LRDs are derived from the general diterpene precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) following two sequential biosynthetic cyclization and/or rearrangement reactions catalyzed by diterpene synthases (diTPSs) [8]. The first step is a protonation-initiated cyclization of GGPP by class II diTPS, mostly by copalyl diphosphate synthase (CPS) that leads to the formation of a bicyclic labdadienyl/copalyl diphosphate (CPP) with a specific stereochemistry (ent, syn, syn-ent or (+)/normal). The most common variant is ent-CPP. Although, (+)- and syn-CPP-producing enzymes were also reported, so far enzyme that produces syn-ent-CPP has not been identified. However, diterpene natural products with syn-ent stereochemistry were recognized from plants of the Calceolaria genus [6]. Beside CPSs, other class II diTPS activities that produce LRDs with endocyclic double bond or oxygen-containing LRDs are also reported [7], [9], [10]. CPP is further cyclised and/or rearranged in a diphosphate ionization-initiated reaction catalyzed by class I diTPS specific to a particular CPP stereoisomer. Subsequent functional modifications of LRDs include addition of hydroxyl group and glycosylation which can be mediated by the activities of cytochrome P450 monooxygenases (CYP450s) and glycosyltransferases (GTs), respectively [11], [12].

To date, major information on LRD biosynthesis has arisen from the biochemical and genetic characterization of the metabolic pathway of ent-kaurene, the precursor of the phytohormone gibberellin (GA) that plays general (i.e. primary) roles in plant growth and development [13], [14]. However, our knowledge of how specialized LRDs are biosynthesised is quite limited, considering a large number of diverse structural variants are already recognized in plants. In angiosperms, ent-kaurene biosynthesis involves two-step cyclization reactions of GGPP that is mediated by two distinct monofunctional diTPSs; a class II diTPS ent-CPP synthase (ent-CPS) and a class I diTPS ent-kaurene synthase (ent-KS), respectively [13], [15]. However, in cases of moss Physcomitrella patens, and fungi such as Gibberella fujikuroi and Phaeosphaeria species L487, a single bifunctional class I/II diTPS (CPS/KS) catalyzes both the cyclization reactions [16], [17], [18]. In contrast to angiosperm where only monofunctional diTPSs are known, gymnosperms have both monofunctional and bifunctional diTPSs. In gymnosperms, the biosynthesis of ent-kaurene destined for the general metabolism is mediated by monofunctional diTPSs; however, several bifunctional class I/II diTPSs with roles in the biosynthesis of specialized LRDs, such as abietadiene synthase (AgAS) of Abies grandis, levopimaradiene synthase of Ginkgo biloba, and isopimaradiene and levopimaradiene/abietadiene synthases of Picea abies and Pinus species, are also known [19], [20], [21], [22].

Despite their distinct reaction mechanisms, bifunctional and monofunctional diTPSs are structurally closely related; comprise of three α helical domains: α, β and γ [23], [24], [25]. The active site of the monofunctional class II diTPSs is located at the interface of β and γ domains and a conserved DxDD general acid motif located at the β domain is responsible for the protonation of C14, C15 double bond of GGPP to initiate cyclization reaction. In cases of monofunctional class I diTPSs, α domain constitutes the active site and two conserved functional motifs, DDxxD and NSE/DTE at the α domain that coordinate three Mg²⁺ ions, trigger ionization-dependent cyclization and/or rearrangement reaction of CPP. While either class I or class II active site is functional in monofunctional diTPSs, both these active sites are functional in bifunctional class I/II diTPSs [26].

Kalmegh (Andrographis paniculata), a medicinal plant of the Acanthaceae family has been widely used in traditional medicine for the treatment of various human health disorders [27]. The utilities of kalmegh as hepatoprotective, anti-inflammatory, anticarcinogenic, anti-HIV, immuno-stimulatory and other human health promoting activities have been mentioned [28], [29]. A number of bioactive specialized metabolites were reported from the leaves and roots of kalmegh, which include ent-LRDs, phenylpropanoids, flavonoids and xanthones [30], [31]. Among these ent-LRDs isolated from leaves such as andrographolide, neoandrographolide and 14-deoxy-11,12-didehydroandrographolide are considered as the main bioactive components [29], [32], [33]. Besides, few other ent-LRDs, both glycosylated and non-glycosylated, are also identified in kalmegh (Fig. S1). These ent-LRDs have similar structural backbones, suggesting sharing of the intermediates during their biosynthesis. Despite effective medicinal utilities of these ent-LRDs, rather limited number of studies have been directed to understand their biosynthesis [34], [35]. The genes/enzymes involved in the biosynthesis of ent-LRDs in kalmegh are yet to be identified and functionally characterized. Earlier, through precursors feeding experiments a major contribution of the methyl erythritol phosphate (MEP) pathway for providing C5 isoprenoids building blocks for the biosynthesis of andrographolide was revealed, although minor contribution from the mevalonate (MEV) pathway was also recognised [34]. Based on known structures of kalmegh ent-LRDs (Fig. S1), we hypothesized that their biosynthesis from GGPP involves at least a class II diTPS similar to ent-CPS, followed by removal of the phosphate group and repeated oxidation of the bicyclic structure that can be mediated by the concerted activities of the class I diTPS/phosphatase and CYP450 enzymes (Fig. 1). The involvement of GT(s) is also expected for the biosynthesis of ent-LRD glycosides.

Here, we report identification and characterization of kalmegh transcripts preferentially expressed in leaf or root tissues, using a suppression subtractive hybridization (SSH) approach. A total of 389 differentially expressed transcripts which represent various specialized metabolic pathways (e.g. terpenoid, phenylpropanoid/flavonoid) were isolated. Further, identification and analysis of two class II monofunctional diTPSs (ApCPS1 and ApCPS2) revealed their involvement in general and specialized metabolisms, respectively. ApCPS1 is likely to be associated with the general metabolism. However, ApCPS2 is potentially involved in tissue-specific accumulation of ent-LRD specialized metabolites. Biochemical characterization of the bacterially expressed recombinant ApCPS2 revealed it to be an ent-CPS that converted GGPP to ent-CPP.