Diterpene synthases of the biosynthetic system of medicinally active diterpenoids in Marrubium vulgare

Abstract

SUMMARYMarrubium vulgare (Lamiaceae) is a medicinal plant whose major bioactive compounds, marrubiin and other labdane-related furanoid diterpenoids, have potential applications as anti-diabetics, analgesics or vasorelaxants. Metabolite and transcriptome profiling of M. vulgare leaves identified five different candidate diterpene synthases (diTPSs) of the TPS-c and TPS-e/f clades. We describe the in vitro and in vivo functional characterization of the M. vulgare diTPS family. In addition to MvEKS ent-kaurene synthase of general metabolism, we identified three diTPSs of specialized metabolism: MvCPS3 (+)-copalyl diphosphate synthase, and the functional diTPS pair MvCPS1 and MvELS. In a sequential reaction, MvCPS1 and MvELS produce a unique oxygenated diterpene scaffold 9,13-epoxy-labd-14-ene en route to marrubiin and an array of related compounds. In contrast with previously known diTPSs that introduce a hydroxyl group at carbon C-8 of the labdane backbone, the MvCPS1-catalyzed reaction proceeds via oxygenation of an intermediate carbocation at C-9, yielding the bicyclic peregrinol diphosphate. MvELS belongs to a subgroup of the diTPS TPS-e/f clade with unusual ba-domain architecture. MvELS is active in vitro and in vivo with three different prenyl diphosphate substrates forming the marrubiin precursor 9,13-epoxy-labd-14-ene, as identified by nuclear magnetic resonance (NMR) analysis, manoyl oxide and miltiradiene. MvELS fills a central position in the biosynthetic system that forms the foundation for the diverse repertoire of Marrubium diterpenoids. Co-expression of MvCPS1 and MvELS in engineered E. coli and Nicotiana benthamiana offers opportunities for producing precursors for an array of biologically active diterpenoids.

Expanding the Landscape of Diterpene Structural Diversity through Stereochemically Controlled Combinatorial Biosynthesis

“…Cf= C. forskohlii ; Ss= S. sclarea ; Mv= M. vulgare ; Ep= Euphorbia peplus ; Os= O. sativa ; Tw= T. wilfordii ; Zm= Z. mays . Diterpenes in samples expressing solely class II diTPSs are likely the result of endogenous phosphatase activity and non‐specific rearrangement (Figure S7) 6a, 7…”

Marrubium vulgare L.: A review on phytochemical and pharmacological aspects

“…By tracer techniques using radiolabeled [3H]-geranylgeranyl diphosphate, confirm that the marrubiin biosynthesis proceeds through the 1-deoxy-D-xylulose-5-phosphate pathway, which was also characterized by gas chromatography with electron impact mass spectrometry technique [46]. Other metabolites and transcription process in M. vulgare for different diterpene synthases (diTPSs), i.e., ent-kaurene synthase, (+)-copalyl diphosphate synthase, and functional diTPS were also identified in leaves [47]. A new secondary metabolites, 11-oxomarrubiin was reported from methanolic extract of whole parts of M. vulgare [48].…”

Functional Diversification of Kaurene Synthase-Like Genes in Isodon rubescens

“…2). This is necessary for proper enzymatic function to produce normal-CPP or normal-LDPP using GGPP as a substrate (Schalk et al, 2012;Pateraki et al, 2014;Zerbe et al, 2014;Cui et al, 2015). The other two genes, IrCPS4 and IrCPS5, have a Trp (W) in this position (Fig.…”

“…These enzymes have the ability to produce abietane, labdane, clerodane, and ent-kaurane diterpenes via an enzymatic hydrolysis-catalyzed reaction (Gao et al, 2009;Cui et al, 2015;Caniard et al, 2012;Schalk et al, 2012;Li et al, 2012;Pateraki et al, 2014;Zerbe et al, 2014;Brückner et al, 2014;Bo zi c et al, 2015;Trikka et al, 2015;Pelot et al, 2017). Based on the stereochemistry of the intermediate that is initially cyclized by CPS, these enzymes can be divided into two groups.…”

“…A similar divergence has also been observed in class I KSL enzymes that are responsible for the subsequent transformation of normal-CPP synthase products. Many of these enzymes have lost the characteristic internal/g domain that is responsible for metabolite specialization, a deletion event found in KSLs in Lamiaceae (Hillwig et al, 2011;Caniard et al, 2012;Schalk et al, 2012;Brückner et al, 2014;Pateraki et al, 2014;Zerbe et al, 2014;Cui et al, 2015;Bo zi c et al, 2015;Trikka et al, 2015). A recent work has shown the plasticity of class I KSLs, such as SsScS from S. sclarea, that can react with any available CPS product (Andersen-Ranberg et al, 2016;Jia et al, 2016).…”