A Cryptic Plant Terpene Cyclase Producing Unconventional 18‐ and 14‐Membered Macrocyclic C₂₅ and C₂₀ Terpenoids with Immunosuppressive Activity

Abstract

Av ersatile terpene synthase (LcTPS2) producing unconventional macrocyclic terpenoids was characterized from Leucosceptrum canum. Engineered Escherichia coli and Nicotiana benthamiana expressing LcTPS2 produced six 18-/14-membered sesterterpenoids including five new ones and two 14-membered diterpenoids.T hese products represent the first macrocyclic sesterterpenoids from plants and the largest sesterterpenoid ring system identified to date.T wo variants F516A and F516G producing approximately 3.3-and 2.5-fold, respectively,m ore sesterterpenoids than the wild-type enzyme were engineered. Both 18-and 14-membered ring sesterterpenoids displayed significant inhibitory activity on the IL-2 and IFN-g production of Tcells probably via inhibition of the MAPK pathway.T he findings will contribute to the development of efficient biocatalysts to create bioactive macrocyclic sesterterpenoids,a nd also herald an ew potential in the welltrodden territory of plant terpenoid biosynthesis.

Turning a Sesquiterpene Synthase into a Di- and Sesterterpene Synthase

“…In contrast, all compounds from LcTPS2, an enzyme that besides 6−8 also produces 4 and 5, uniformly show a 14S configuration. 27 In order to confirm our results, (R)-and (S)-(1-13 C,1-2 H)GGPP 28 were incubated with SvES-F77G, resulting in the stereoselective incorporation of deuterium into the diastereotopic hydrogens at C-1 of 4 and 5 (Figure S12). In the analogous experiment with (R)-and (S)-(1-13 C,1-2 H)GFPP 29 and SvES-F77A, also a stereoselective incorporation into the hydrogens at C-1 of 6 and 8 was observed, but in each experiment into the other of the two diastereotopic hydrogen atoms, which confirms the different C-14 configurations for 4 and 5 versus 6 and 8.…”

“…Nevertheless, its structure was confirmed by comparison of its 1 H NMR spectrum and the EI mass spectrum to published data. 27 All three sesterterpenes 6−8 were previously obtained from the plant terpene synthase LcTPS2 from Leucosceptrum canum. 27 The optical rotation of 6 ([α] D 25 = −8.7, c 0.05, CH 2 Cl 2 ) pointed to the same enantiomer as for (2E,14S)-cericerene obtained with LcTPS2 ([α] D 25 = −5.94, c 0.33, CHCl 3 ), and analogously also for 8, the same enantiomer as for the compound from LcTPS2 can be concluded ([α] D 25 = +75.0, c 0.01, CH 2 Cl 2 ; lit.…”

“…27 The optical rotation of 6 ([α] D 25 = −8.7, c 0.05, CH 2 Cl 2 ) pointed to the same enantiomer as for (2E,14S)-cericerene obtained with LcTPS2 ([α] D 25 = −5.94, c 0.33, CHCl 3 ), and analogously also for 8, the same enantiomer as for the compound from LcTPS2 can be concluded ([α] D 25 = +75.0, c 0.01, CH 2 Cl 2 ; lit. : 27 [α] D 25 = +106.7, c 0.04, CHCl 3 ). Interestingly, (2E,14S)-6 was recently also reported from a sextuple variant of the diterpene synthase VenA.…”

Two Sesterterpene Synthases from Lentzea atacamensis Demonstrate the Role of Conformational Variability in Terpene Biosynthesis

“…[29] In contrast, sesterterpene assembly in plants is carried out by two discrete enzymes, a GFPP synthase (GFPPS) and a StTS, that are often genetically clustered. [30][31][32][33] Also in bacteria sesterterpene biosynthesis is catalyzed by two independent GFPPS and StTS enzymes. Besides the UbiA-related StTS StsC from Streptomyces somaliensis for somaliensenes A and B [34] and the type II StTS AtoE from Amycolatopsis tolypomycina involved in the biosynthesis of atolypenes A and B, [35] only four bacterial type I StTSs have been reported so far.…”

Zwei Sesterterpen‐Syntasen aus Lentzea atacamensis demonstrieren die Rolle der Konformationsvariabilität in der Terpenbiosynthese

“…[29] Im Gegensatz dazu erfolgt der Sesterterpenaufbau in Pflanzen durch zwei separate Enzyme, eine GFPP-Synthase (GFPPS) und eine StTS, die oft genetisch geclustert sind. [30][31][32][33] Auch in Bakterien wird die Sesterterpen-Biosynthese durch zwei unabhängige GFPPS und StTS katalysiert. Neben der UbiAverwandten StTS StsC aus Streptomyces somaliensis für die Somaliensene A und B [34] und der Typ-II-StTS AtoE aus Amycolatopsis tolypomycina, das an der Biosynthese der Atolypene A und B beteiligt ist, [35] wurden bisher nur vier bakterielle Typ-I-StTS beschrieben.…”