Jan Rinkel

Conformational Analysis, Thermal Rearrangement, and EI-MS Fragmentation Mechanism of (1(10)E,4E,6S,7R)-Germacradien-6-ol by (13)C-Labeling Experiments.

An uncharacterized terpene cyclase from Streptomyces pratensis was identified as (+)-(1(10)E,4E,6S,7R)-germacradien-6-ol synthase. The enzyme product exists as two interconvertible conformers, resulting in complex NMR spectra. For the complete assignment of NMR data, all fifteen ((13)C1)FPP isotopomers (FPP=farnesyl diphosphate) and ((13)C15)FPP were synthesized and enzymatically converted. The products were analyzed using various NMR techniques, including (13)C, (13)C COSY experiments. The ((13)C)FPP isotopomers were also used to investigate the thermal rearrangement and EI fragmentation of the enzyme product.

Mechanistic Investigations of Two Bacterial Diterpene Cyclases: Spiroviolene Synthase and Tsukubadiene Synthase

The mechanisms of two diterpene cyclases from streptomycetes-one with an unknown product that was identified as the spirocyclic hydrocarbon spiroviolene and one with the known product tsukubadiene-were investigated in detail by isotope labeling experiments. Although the structures of the products were very different, the cyclization mechanisms of both enzymes proceed through the same initial cyclization reactions, before they diverge towards the individual products, which is reflected in the close phylogenetic relationship of the enzymes.

Recent highlights in biosynthesis research using stable isotopes

Summary The long and successful history of isotopic labeling experiments within natural products research has both changed and deepened our understanding of biosynthesis. As demonstrated in this article, the usage of isotopes is not at all old-fashioned, but continues to give important insights into biosynthetic pathways of secondary metabolites. This review with 85 cited references is structured by separate discussions of compounds from different classes including polyketides, non-ribosomal peptides, their hybrids, terpenoids, and aromatic compounds formed via the shikimate pathway. The text does not aim at a comprehensive overview, but instead a selection of recent important examples of isotope usage within biosynthetic studies is presented, with a special emphasis on mechanistic surprises.

Lessons from 1,3-Hydride Shifts in Sesquiterpene Cyclizations.

Stereospecifically labelled precursors were subjected to conversion by seven bacterial sesquiterpene cyclases to investigate the stereochemistry of their initial 1,10-cyclisation-1,3-hydride shift cascades. Enzymes with products of known absolute configuration showed a coherent stereochemical course, except for (-)-α-amorphene synthase, for which the obtained results are better explained by an initial 1,6-cyclisation. The link between the absolute configuration of the product and the stereochemical course of the 1,3-hydride shifts enabled assignment of the absolute configurations of three enzyme products, which were confirmed independently through the absolute configuration of the common byproduct germacrene D-4-ol.

Terpene cyclases from social Amoebae

Genome sequences of social amoebae reveal the presence of terpene cyclases (TCs) in these organisms. Two TCs from Dictyostelium discoideum converted farnesyl diphosphate into (2S,3R,6S,9S)-(-)-protoillud-7-ene and (3S)-(+)-asterisca-2(9),6-diene. The enzyme mechanisms and EI-MS fragmentations of the products were studied by labeling experiments.

Mechanistic Characterization of Two Chimeric Sesterterpene Synthases from Penicillium

The products of two bifunctional fungal sesterterpene synthases (StTPS), with prenyl transferase (PT) and terpene synthase (TPS) domains from Penicillium, were structurally characterized and their mechanisms studied in detail by labeling experiments. A phylogenetic analysis of the TPS domains of the new and previously characterized enzymes revealed six distinct clades. Enzymes from the same clade catalyze a common initial cyclization step, which suggests the potential for structural predictions from amino acid sequences.

Mechanisms of the Diterpene Cyclases β‐Pinacene Synthase from Dictyostelium discoideum and Hydropyrene Synthase from Streptomyces clavuligerus

Two diterpene cyclases, one from the social amoeba Dictyostelium discoideum and the other from the bacterium Streptomyces clavuligerus, with products containing a Z-configured double bond between the original C2 and C3 of geranylgeranyl diphosphate, were extensively investigated for their mechanisms through isotopic labelling experiments. The participation of geranyllinalyl diphosphate, in analogy to the role of linalyl and nerolidyl diphosphate for mono- and sesquiterpene biosynthesis, as an intermediate towards diterpenes with a Z-configured C2=C3 double bond is discussed.

Spata-13,17-diene Synthase, an Enzyme with Sesqui-, Di- and Sesterterpene Synthase Activity from Streptomyces xinghaiensis

A terpene synthase from the marine bacterium Streptomyces xinghaiensis has been characterised, including a full structure elucidation of its products from various substrates and an in-depth investigation of the enzyme mechanism by isotope labelling experiments, metal cofactor variations, and mutation experiments. The results revealed an interesting dependency of Mn2+ catalysis on the presence of Asp-217, a residue that is occupied by a highly conserved Glu in most other bacterial terpene synthases.

Two Diterpene Synthases for Spiroalbatene and Cembrene A from Allokutzneria albata

Two bacterial diterpene synthases from the actinomycete Allokutzneria albata were investigated, resulting in the identification of the structurally unprecedented compound spiroalbatene from the first and cembrene A from the second enzyme. Both enzymes were thoroughly investigated in terms of their mechanisms by isotope labeling experiments, site-directed mutagenesis, and variation of the metal cofactors and pH value. For spiroalbatene synthase, the pH- and Mn2+ -dependent formation of the side product thunbergol was observed, which is biosynthetically linked to spiroalbatene.

18-Hydroxydolabella-3,7-diene synthase - a diterpene synthase from Chitinophaga pinensis

The product obtained in vitro from a diterpene synthase encoded in the genome of the bacterium Chitinophaga pinensis, an enzyme previously reported to have germacrene A synthase activity during heterologous expression in Escherichia coli, was identified by extensive NMR-spectroscopic methods as 18-hydroxydolabella-3,7-diene. The absolute configuration of this diterpene alcohol and the stereochemical course of the terpene synthase reaction were addressed by isotopic labelling experiments. Heterologous expression of the diterpene synthase in Nicotiana benthamiana resulted in the production of 18-hydroxydolabella-3,7-diene also in planta, while the results from the heterologous expression in E. coli were shown to be reproducible, revealing that the expression of one and the same terpene synthase in different heterologous hosts may yield different terpene products.