Bifunctional cis-Abienol Synthase from Abies balsamea Discovered by Transcriptome Sequencing and Its Implications for Diterpenoid Fragrance Production

Zerbe, Philipp; Chiang, Angela; Yuen, Macaire Man Saint; Hamberger, Bjoern; Hamberger, Britta; Draper, Jason A.; Britton, Robert; Bohlmann, Jöerg

doi:10.1074/jbc.m111.317669

Cited by 76 publications

(104 citation statements)

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“…Before this study, knowledge of secondary diterpene metabolism in gymnosperms included the bifunctional class I/II diTPSs involved in the biosynthesis of labdane diterpenes, derived diterpene resin acids of conifer chemical defense (16,38), and a few monofunctional class I diTPSs involved in diterpene resin acid biosynthesis in pine and taxane formation in species of yew (Fig. S1) (17,18).…”

Section: Discussionmentioning

confidence: 99%

Biosynthesis of the microtubule-destabilizing diterpene pseudolaric acid B from golden larch involves an unusual diterpene synthase

Mafu

Karunanithi

Palazzo

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The diversity of small molecules formed via plant diterpene metabolism offers a rich source of known and potentially new biopharmaceuticals. Among these, the microtubule-destabilizing activity of pseudolaric acid B (PAB) holds promise for new anticancer agents. PAB is found, perhaps uniquely, in the coniferous tree golden larch (Pseudolarix amabilis, Pxa). Here we describe the discovery and mechanistic analysis of golden larch terpene synthase 8 (PxaTPS8), an unusual diterpene synthase (diTPS) that catalyzes the first committed step in PAB biosynthesis. Mining of the golden larch root transcriptome revealed a large TPS family, including the monofunctional class I diTPS PxaTPS8, which converts geranylgeranyl diphosphate into a previously unknown 5,7-fused bicyclic diterpene, coined "pseudolaratriene." Combined NMR and quantum chemical analysis verified the structure of pseudolaratriene, and co-occurrence with PxaTPS8 and PAB in P. amabilis tissues supports the intermediacy of pseudolaratriene in PAB metabolism. Although PxaTPS8 adopts the typical three-domain structure of diTPSs, sequence phylogeny places the enzyme with two-domain TPSs of mono-and sesqui-terpene biosynthesis. Site-directed mutagenesis of PxaTPS8 revealed several catalytic residues that, together with quantum chemical calculations, suggested a substantial divergence of PxaTPS8 from other TPSs leading to a distinct carbocation-driven reaction mechanism en route to the 5,7-trans-fused bicyclic pseudolaratriene scaffold. PxaTPS8 expression in microbial and plant hosts provided proof of concept for metabolic engineering of pseudolaratriene. diterpene biosynthesis | Pseudolarix amabilis | plant natural products | pseudolaric acid | chemotherapeutic drug

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Section: Discussionmentioning

confidence: 99%

Biosynthesis of the microtubule-destabilizing diterpene pseudolaric acid B from golden larch involves an unusual diterpene synthase

Mafu

Karunanithi

Palazzo

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…S1) to produce various bicyclic or, more commonly, tricyclic diterpene structures (Peters, 2010;Zerbe and Bohlmann, 2015a). Families of diTPSs, including the isopimaradiene synthases (ISO) and levopimaradiene/abietadiene synthases (LAS), have been characterized in several spruce and pine species (Martin et al, 2004;Ro and Bohlmann, 2006;Keeling et al, 2011;Zerbe et al, 2012;Hall et al, 2013b). A general picture of conifer diTPSs has emerged, with the majority representing bifunctional class I/II enzymes with two active sites (Zhou et al, 2012;Zerbe and Bohlmann, 2015b).…”

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Modularity of Conifer Diterpene Resin Acid Biosynthesis: P450 Enzymes of Different CYP720B Clades Use Alternative Substrates and Converge on the Same Products

et al. 2016

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Cytochrome P450 enzymes of the CYP720B subfamily play a central role in the biosynthesis of diterpene resin acids (DRAs), which are a major component of the conifer oleoresin defense system. CYP720Bs exist in families of up to a dozen different members in conifer genomes and fall into four different clades (I-IV). Only two CYP720B members, loblolly pine (Pinus taeda) PtCYP720B1 and Sitka spruce (Picea sitchensis) PsCYP720B4, have been characterized previously. Both are multisubstrate and multifunctional clade III enzymes, which catalyze consecutive three-step oxidations in the conversion of diterpene olefins to DRAs. These reactions resemble the sequential diterpene oxidations affording ent-kaurenoic acid from ent-kaurene in gibberellin biosynthesis. Here, we functionally characterized the CYP720B clade I enzymes CYP720B2 and CYP720B12 in three different conifer species, Sitka spruce, lodgepole pine (Pinus contorta), and jack pine (Pinus banksiana), and compared their activities with those of the clade III enzymes CYP720B1 and CYP720B4 of the same species. Unlike the clade III enzymes, clade I enzymes were ultimately found not to be active with diterpene olefins but converted the recently discovered, unstable diterpene synthase product 13-hydroxy-8(14)-abietene. Through alternative routes, CYP720B enzymes of both clades produce some of the same profiles of conifer oleoresin DRAs (abietic acid, neoabietic acid, levopimaric acid, and palustric acid), while clade III enzymes also function in the formation of pimaric acid, isopimaric acid, and sandaracopimaric acid. These results highlight the modularity of the specialized (i.e. secondary) diterpene metabolism, which produces conifer defense metabolites through variable combinations of different diterpene synthase and CYP720B enzymes.

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“…As with the class II diTPSs, deprotonation or water capture terminate the class I diTPS reaction. Water capture as a mechanism of diTPSs that leads to oxygen functionalities in the diterpene products has been described for the class II active site of bifunctional class I/II gymnosperm diTPSs (Keeling et al, 2011;Zerbe et al, 2012Zerbe et al, , 2013, class II angiosperm diTPSs (Falara et al, 2010;Caniard et al, 2012;Zerbe et al, 2013), and class I angiosperm diTPS (Caniard et al, 2012). Further oxidative functionalization of diterpenoids is typically catalyzed by cytochrome P450-dependent monooxygenase enzymes (P450s; Ro et al, 2005;Swaminathan et al, 2009;Hamberger et al, 2011;Wang et al, 2011;Guo et al, 2013;Hamberger and Bak, 2013), which then provide molecular handles for addition of auxiliary functional groups, leading ultimately to highly complex and decorated products such as forskolin and its derivatives.…”

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confidence: 99%

Manoyl Oxide (13R), the Biosynthetic Precursor of Forskolin, Is Synthesized in Specialized Root Cork Cells in Coleus forskohlii

et al. 2014

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Bifunctional cis-Abienol Synthase from Abies balsamea Discovered by Transcriptome Sequencing and Its Implications for Diterpenoid Fragrance Production

Cited by 76 publications

References 56 publications

Biosynthesis of the microtubule-destabilizing diterpene pseudolaric acid B from golden larch involves an unusual diterpene synthase

Biosynthesis of the microtubule-destabilizing diterpene pseudolaric acid B from golden larch involves an unusual diterpene synthase

Modularity of Conifer Diterpene Resin Acid Biosynthesis: P450 Enzymes of Different CYP720B Clades Use Alternative Substrates and Converge on the Same Products

Manoyl Oxide (13R), the Biosynthetic Precursor of Forskolin, Is Synthesized in Specialized Root Cork Cells in Coleus forskohlii

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