Patrick Rabe

Induced-fit mechanism in class I terpene cyclases.

We present crystallographic and functional data of selina-4(15),7(11)-diene synthase (SdS) from Streptomyces pristinaespiralis in its open and closed (ligand-bound) conformation. We could identify an induced-fit mechanism by elucidating a rearrangement of the G1/2 helix-break motif upon substrate binding. This rearrangement highlights a novel effector triad comprising the pyrophosphate sensor Arg178, the linker Asp181, and the effector Gly182-O. This structural motif is strictly conserved in class I terpene cyclases from bacteria, fungi, and plants, including epi-isozizaene synthase (3KB9), aristolochene synthase (4KUX), bornyl diphosphate synthase (1N20), limonene synthase (2ONG), 5-epi-aristolochene synthase (5EAT), and taxa-4(5),11(12)-diene synthase (3P5R). An elaborate structure-based mutagenesis in combination with analysis of the distinct product spectra confirmed the mechanistic models of carbocation formation and stabilization in SdS.

The Scent of Bacteria: Headspace Analysis for the Discovery of Natural Products

Volatile compounds released by 50 bacterial strains, 45 of them actinobacteria in addition to three chloroflexi and two myxobacteria, have been collected by use of a closed-loop stripping apparatus, and the obtained headspace extracts have been analyzed by GC-MS. Excluding terpenes that have recently been published elsewhere, 254 compounds from all kinds of compound classes have been identified. For unambiguous compound identification several reference compounds have been synthesized. Among the detected volatiles 12 new natural products have been found, in addition to mellein, which was released by Saccharopolyspora erythraea. The iterative PKS for this compound has recently been identified by in vitro experiments, but mellein production in S. erythraea has never been reported before. These examples demonstrate that headspace analysis is an important tool for the discovery of natural products that may be overlooked using conventional techniques. The method is also useful for feeding experiments with isotopically labeled precursors and was applied to investigate the biosynthesis of the unusual nitrogen compound 1-nitro-2-methylpropane, which arises from valine. Furthermore, several streptomycetes emitted compounds that were previously recognized as insect pheromones, thus questioning if bacterial symbionts are involved in insect communication.

Hedycaryol synthase in complex with nerolidol reveals terpene cyclase mechanism.

The biosynthesis of terpenes is catalysed by class I and II terpene cyclases. Here we present structural data from a class I hedycaryol synthase in complex with nerolidol, serving as a surrogate for the reaction intermediate nerolidyl diphosphate. This prefolded ligand allows mapping of the active site and hence the identification of a key carbonyl oxygen of Val179, a highly conserved helix break (G1/2) and its corresponding helix dipole. Stabilising the carbocation at the substrates C1 position, these elements act in concert to catalyse the 1,10 ring closure, thereby exclusively generating the anti‐Markovnikov product. The delineation of a general mechanistic scaffold was confirmed by site‐specific mutations. This work serves as a basis for understanding carbocation chemistry in enzymatic reactions and should contribute to future application of these enzymes in organic synthesis.

An Improved Technique for the Rapid Chemical Characterisation of Bacterial Terpene Cyclases

A derivative of the pET28c(+) expression vector was constructed. It contains a yeast replication system (2μ origin of replication) and a yeast selectable marker (URA3), and can be used for gene cloning in yeast by efficient homologous recombination, and for heterologous expression in E. coli. The vector was used for the expression and chemical characterisation of three bacterial terpene cyclases.

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.

Volatile terpenes from actinomycetes: a biosynthetic study correlating chemical analyses to genome data.

The volatile terpenes of 24 actinomycetes whose genomes have been sequenced (or are currently being sequenced) were collected by use of a closed‐loop stripping apparatus and identified by GC/MS. The analytical data were compared against a phylogenetic analysis of all 192 currently available sequences of bacterial terpene cyclases (excluding geosmin and 2‐methylisoborneol synthases). In addition to the several groups of terpenes with known biosynthetic origin, selinadienes were identified as a large group of biosynthetically related sesquiterpenes that are produced by several streptomycetes. The detection of a large number of previously unrecognised side products of known terpene cyclases proved to be particularly important for an in depth understanding of biosynthetic pathways to known terpenes in actinomycetes. Interpretation of the chemical analytical data in the context of the phylogenetic tree of bacterial terpene cyclases pointed to the function of three new enzymes: (E)‐β‐caryophyllene synthase, selina‐3,7(11)‐diene synthase and aristolochene synthase.

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.

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.

Structures and Biosynthesis of Corvol Ethers--Sesquiterpenes from the Actinomycete Kitasatospora setae.

Here we present the functional characterization of a sesquiterpene cyclase from Kitasatospora setae. The enzyme converts the sesquiterpene precursor farnesyl diphosphate (FPP) into two previously unknown and unstable sesquiterpene ethers for which we propose the trivial names corvol ethers A and B. Both compounds were purified and their structures were determined by one- and two-dimensional NMR spectroscopy. A biosynthetic mechanism for the FPP cyclization by the corvol ether synthase was proposed. The results from the incubation experiments of the corvol ether synthase with isotopically labeled precursors were in line with this mechanism, while alternative mechanisms could clearly be ruled out.

Terpene synthase genes in eukaryotes beyond plants and fungi: Occurrence in social amoebae

Significance Many living organisms use terpenes for ecological interactions. Terpenes are biosynthesized by terpene synthases (TPSs), but classic TPS genes are known to exist only in plants and fungi among the eukaryotes. In this study, TPS genes were identified in six species of amoebae with five of them being multicellular social amoebae. Amoebal TPSs showed closer relatedness to fungal TPSs than bacterial TPSs. In the social amoeba Dictyostelium discoideum, all nine TPS genes encoded active enzymes and most of their terpene products were released as volatiles in a development-specific manner. This study highlights a wider distribution of TPS genes in eukaryotes than previously thought and opens a door to studying the function and evolution of TPS genes and their products. Terpenes are structurally diverse natural products involved in many ecological interactions. The pivotal enzymes for terpene biosynthesis, terpene synthases (TPSs), had been described only in plants and fungi in the eukaryotic domain. In this report, we systematically analyzed the genome sequences of a broad range of nonplant/nonfungus eukaryotes and identified putative TPS genes in six species of amoebae, five of which are multicellular social amoebae from the order of Dictyosteliida. A phylogenetic analysis revealed that amoebal TPSs are evolutionarily more closely related to fungal TPSs than to bacterial TPSs. The social amoeba Dictyostelium discoideum was selected for functional study of the identified TPSs. D. discoideum grows as a unicellular organism when food is abundant and switches from vegetative growth to multicellular development upon starvation. We found that expression of most D. discoideum TPS genes was induced during development. Upon heterologous expression, all nine TPSs from D. discoideum showed sesquiterpene synthase activities. Some also exhibited monoterpene and/or diterpene synthase activities. Direct measurement of volatile terpenes in cultures of D. discoideum revealed essentially no emission at an early stage of development. In contrast, a bouquet of terpenes, dominated by sesquiterpenes including β-barbatene and (E,E)-α-farnesene, was detected at the middle and late stages of development, suggesting a development-specific function of volatile terpenes in D. discoideum. The patchy distribution of TPS genes in the eukaryotic domain and the evidence for TPS function in D. discoideum indicate that the TPS genes mediate lineage-specific adaptations.