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

Abstract

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

Protonation‐Initiated Cyclization by a Class II Terpene Cyclase Assisted by Tunneling

“…Subsequent cyclization of these relatively simple, linear building blocks by terpene cyclases enables the generation of a vast array of multicyclic molecular architectures, with potent biological activities . Stereospecific generation of tailored polycyclic terpenes provides access to chiral fine chemical synthons and antifungal, ‐viral, ‐microbial, and ‐cancer agents . Bifunctional terpene synthases with distinct domains for linear terpene formation and cyclization have been described .…”

Oleoresin defenses in conifers: chemical diversity, terpene synthases and limitations of oleoresin defense under climate change

“…Beyond oleoresin and gibberellin biosynthesis, products of various conifer di‐TPSs and their derivatives are of interest as high‐value bioproducts, including pharmaceuticals (Zerbe et al ., ). Example are the anticancer drug taxol found in yew (Croteau et al ., ), the antitumor compound pseudolaric acid B produced in the roots of Pseudolarix amabilis (Mafu et al ., ), and c is ‐abienol as a molecule of interest in the fragrance industry produced in balsam fir (Zerbe et al ., ).…”

Biochemical characterization of diterpene synthases of Taiwania cryptomerioides expands the known functional space of specialized diterpene metabolism in gymnosperms

“…Pairwise activity of the remaining class‐II and class‐I diTPSs resulted in the formation of a range of specialized diterpenoids, including manoyl oxide derived from LPP ( Tc KSL1 and Tc KSL3‐7), as well as the conversion of (+)‐CPP into biformene ( Tc KSL1), levopimaradiene ( Tc KSL3) and phyllocladanol ( Tc KSL5), as verified by NMR analysis and authentic standards. Although levopimaradiene formation by Tc CPS4 and Tc KSL3 is functionally analogous to bifunctional class‐I/II diTPSs producing levopimaradiene in other gymnosperm species (Peters et al ., ; Schepmann et al ., ; Martin et al ., ; Hall et al ., ; Mafu et al ., ; Celedon and Bohlmann, ), diTPSs forming phyllocladanol or biformene have not previously been reported in gymnosperms. Notably, abienol synthase from Nicotiana tabacum has been shown to produce biformene when acting together with a (+)‐CPS (Sallaud et al ., ; Ignea et al ., ).…”

“…Although the presence of class‐II/I diTPSs in T. cryptomerioides cannot be excluded, given that five of the transcripts identified could only be obtained as partial sequences, it appears that diterpenoid metabolism in T. cryptomerioides relies predominantly, or perhaps exclusively, on pairwise‐acting monofunctional diTPSs (Figure ). Thus, the diTPSs identified in this study add new catalysts to the group of monofunctional class‐I diTPSs involved in gymnosperm specialized metabolism, which also includes taxadiene synthases in species of yew ( Taxus ) that directly convert GGPP into the macrocyclic intermediate taxadiene and related compounds (Wildung and Croteau, ), monofunctional pimaradiene and isopimaradiene synthases involved in DRA biosynthesis in Jack pine and lodgepole pine (Hall et al ., ), and the pseudolaratriene synthase of Pseudolarix amabilis (golden larch) that converts GGPP into the unusual bicyclic diterpene pseudolaratriene (Mafu et al ., ).…”