The Diterpenoid Substrate Analogue 19-nor-GGPP Reveals Pronounced Methyl Group Effects in Diterpene Cyclisations.

The substrate analogue 19-nor-geranylgeranyl diphosphate (19-nor-GGPP) was synthesised and incubated with 20 diterpene synthases, resulting in the formation of diterpenoids in all cases. A total of 23 different compounds were isolated from these enzyme reactions and structurally characterised, if possible including the experimental determination of absolute configurations through a stereoselective deuteration approach. In several cases the missing 19-Me group in the substrate analogue resulted in opening of completely new reaction paths towards compounds with novel skeletons. DFT calculations were applied to gain a deeper understanding of these observed methyl group effects in diterpene biosynthesis.

Spiroluchuene A Synthase: A Cyclase from Aspergillus luchuensis Forming a Spirotetracyclic Diterpene.

The diterpene synthase AlTS was identified from Aspergillus luchuensis. AlTS catalyses the formation of the diterpene hydrocarbon spiroluchuene A, which exhibits a novel skeleton characterised by a spirocyclic ring system. The cyclisation mechanism towards this compound was elucidated through isotopic labelling experiments in conjunction with DFT calculations and metadynamic simulations. The biosynthetic intermediate luchudiene, besides the derivative spiroluchuene B, was captured from an enzyme variant obtained through site-directed mutagenesis. With its 10-membered ring luchudiene is structurally related to germacrenes and can undergo a Cope rearrangement to luchuelemene.

Functional characterisation of twelve terpene synthases from actinobacteria.

Fifteen type I terpene synthase homologs from diverse actinobacteria that were selected based on a phylogenetic analysis of more than 4000 amino acid sequences were investigated for their products. For four enzymes with functions not previously reported from bacterial terpene synthases the products were isolated and their structures were elucidated by NMR spectroscopy, resulting in the discovery of the first terpene synthases for (+)-δ-cadinol and (+)-α-cadinene, besides the first two bacterial (-)-amorpha-4,11-diene synthases. For other terpene synthases with functions reported from bacteria before the products were identified by GC-MS. The characterised enzymes include a new epi-isozizaene synthase with monoterpene synthase side activity, a 7-epi-α-eudesmol synthase that also produces hedycaryol and germacrene A, and four more sesquiterpene synthases that produce mixtures of hedycaryol and germacrene A. Three phylogenetically related enzymes were in one case not expressed and in two cases inactive, suggesting pseudogenisation in the respective branch of the phylogenetic tree. Furthermore, a diterpene synthase for allokutznerene and a sesterterpene synthase for sesterviolene were identified.

Mechanistic Characterisation of Collinodiene Synthase, a Diterpene Synthase from Streptomyces collinus.

Two homologs of the diterpene synthase CotB2 from Streptomyces collinus (ScCotB2) and Streptomyces iakyrus (SiCotB2) were investigated for their products by in vitro incubations of the recombinant enzymes with geranylgeranyl pyrophosphate, followed by compound isolation and structure elucidation by NMR. ScCotB2 produced the new compound collinodiene, besides the canonical CotB2 product cyclooctat-9-en-7-ol, dolabella-3,7,18-triene and dolabella-3,7,12-triene, while SiCotB2 gave mainly cyclooctat-9-en-7-ol and only traces of dolabella-3,7,18-triene. The cyclisation mechanism towards the ScCotB2 products and their absolute configurations were investigated through isotopic labelling experiments.