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.

The chemical biology of dimethylsulfoniopropionate.

Dimethylsulfoniopropionate is a highly abundant sulfur metabolite in marine ecosystems. Its biosynthesis by different organisms including plants, marine algae and dinoflagellates is discussed. Furthermore, the accumulated knowledge about bacterial uptake systems and its climatically relevant degradation by marine bacteria to methanethiol or dimethylsulfide is presented. Finally, uptake and degradation of synthetic DMSP analogs are addressed.

Volatiles from nineteen recently genome sequenced actinomycetes.

The volatiles released by agar plate cultures of nineteen actinomycetes whose genomes were recently sequenced were collected by use of a closed-loop stripping apparatus (CLSA) and analysed by GC/MS. In total, 178 compounds from various classes were identified. The most interesting findings were the detection of the insect pheromone frontalin in Streptomyces varsoviensis, and the emission of the unusual plant metabolite 1-nitro-2-phenylethane. Its biosynthesis from phenylalanine was investigated in isotopic labelling experiments. Furthermore, the identified terpenes were correlated to the information about terpene cyclase homologs encoded in the investigated strains. The analytical data were in line with functionally characterised bacterial terpene cyclases and particularly corroborated the recently suggested function of a terpene cyclase from Streptomyces violaceusniger by the identification of a functional homolog in Streptomyces rapamycinicus.

Synthesis and bioactivity of analogues of the marine antibiotic tropodithietic acid.

Tropodithietic acid (TDA) is a structurally unique sulfur-containing antibiotic from the Roseobacter clade bacterium Phaeobacter inhibens DSM 17395 and a few other related species. We have synthesised several structural analogues of TDA and used them in bioactivity tests against Staphylococcus aureus and Vibrio anguillarum for a structure-activity relationship (SAR) study, revealing that the sulfur-free analogue of TDA, tropone-2-carboxylic acid, has an antibiotic activity that is even stronger than the bioactivity of the natural product. The synthesis of this compound and of several analogues is presented and the bioactivity of the synthetic compounds is discussed.

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.

Identification of isoafricanol and its terpene cyclase in Streptomyces violaceusniger using CLSA-NMR.

The recently developed CLSA-NMR technique that is based on feeding experiments with (13)C-labelled precursors was applied in the identification of isoafricanol as the main volatile terpene emitted by Streptomyces violaceusniger. The isoafricanol synthase of this organism is presented, together with a recent phylogenetic analysis of bacterial terpene cyclases.

A volatile lactone of Hymenoscyphus pseudoalbidus, pathogen of European ash dieback, inhibits host germination.

The largely unknown secondary metabolism of the plant pathogenic fungus Hymenoscyphus pseudoalbidus was investigated by use of the CLSA method. A set of volatile lactones was identified by GC/MS. The lactones were synthesized and used in bioassays in which one of the compounds was found to be a strong germination inhibitor for ash seeds, causing necroses in the plant tissue.

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 substrate's 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.

Labelling studies on the biosynthesis of terpenes in Fusarium fujikuroi.

Synthetic [2-(13)C]mevalonolactone was fed to the gibberellin producer Fusarium fujikuroi and its incorporation into four known terpenoids was investigated by (13)C NMR analysis of crude culture extracts. The experiments gave detailed insights into the mechanisms of terpene biosynthesis by this fungus.

Halogenated volatiles from the fungus Geniculosporium and the actinomycete Streptomyces chartreusis.

Two unidentified chlorinated volatiles X and Y were detected in headspace extracts of the fungus Geniculosporium. Their mass spectra pointed to the structures of a chlorodimethoxybenzene for X and a dichlorodimethoxybenzene for Y. The mass spectra of some constitutional isomers for X and Y were included in our databases and proved to be very similar, thus preventing a full structural assignment. For unambiguous structure elucidation all possible constitutional isomers for X and Y were obtained by synthesis or from commercial suppliers. Comparison of mass spectra and GC retention times rigorously established the structures of the two chlorinated volatiles. Chlorinated volatiles are not very widespread, but brominated or even iodinated volatiles are even more rare. Surprisingly, headspace extracts from Streptomyces chartreusis contained methyl 2-iodobenzoate, a new natural product that adds to the small family of iodinated natural products.

[(2)H26]-1-epi-Cubenol, a completely deuterated natural product from Streptomyces griseus.

During growth on fully deuterated medium the volatile terpene [(2)H26]-1-epi-cubenol was released by the actinomycete Streptomyces griseus. This compound represents the first completely deuterated terpene obtained by fermentation. Despite a few previous reports in the literature the operability of this approach to fully deuterated compounds is still surprising, because the strong kinetic isotope effect of deuterium is known to slow down all metabolic processes in living organisms. Potential applications of completely labelled compounds from natural sources in structure elucidation, biosynthetic or pharmacokinetic investigations are discussed.

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)-ß-caryophyllene synthase, selina-3,7(11)-diene synthase and aristolochene synthase.

The stereochemical course of tricho-acorenol biosynthesis.

The biosynthesis of tricho-acorenol in Trichoderma harzianum was investigated by feeding stereospecifically deuterated mevalonolactone isotopomers, followed by a detailed GC/MS analysis of the incorporation of labelling. The results establish a highly stereospecific hydride migration and antarafacial attack of water in the terminal step towards tricho-acorenol.

Isotopically labeled sulfur compounds and synthetic selenium and tellurium analogues to study sulfur metabolism in marine bacteria.

Members of the marine Roseobacter clade can degrade dimethylsulfoniopropionate (DMSP) via competing pathways releasing either methanethiol (MeSH) or dimethyl sulfide (DMS). Deuterium-labeled [(2)H6]DMSP and the synthetic DMSP analogue dimethyltelluriopropionate (DMTeP) were used in feeding experiments with the Roseobacter clade members Phaeobacter gallaeciensis DSM 17395 and Ruegeria pomeroyi DSS-3, and their volatile metabolites were analyzed by closed-loop stripping and solid-phase microextraction coupled to GC-MS. Feeding experiments with [(2)H6]DMSP resulted in the incorporation of a deuterium label into MeSH and DMS. Knockout of relevant genes from the known DMSP demethylation pathway to MeSH showed in both species a residual production of [(2)H3]MeSH, suggesting that a second demethylation pathway is active. The role of DMSP degradation pathways for MeSH and DMS formation was further investigated by using the synthetic analogue DMTeP as a probe in feeding experiments with the wild-type strain and knockout mutants. Feeding of DMTeP to the R. pomeroyi knockout mutant resulted in a diminished, but not abolished production of demethylation pathway products. These results further corroborated the proposed second demethylation activity in R. pomeroyi. Isotopically labeled [(2)H3]methionine and (34)SO4 (2-), synthesized from elemental (34)S8, were tested to identify alternative sulfur sources besides DMSP for the MeSH production in P. gallaeciensis. Methionine proved to be a viable sulfur source for the MeSH volatiles, whereas incorporation of labeling from sulfate was not observed. Moreover, the utilization of selenite and selenate salts by marine alphaproteobacteria for the production of methylated selenium volatiles was explored and resulted in the production of numerous methaneselenol-derived volatiles via reduction and methylation. The pathway of selenate/selenite reduction, however, proved to be strictly separated from sulfate reduction.

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.

Terpenoids are widespread in actinomycetes: a correlation of secondary metabolism and genome data.

The genomes of all bacteria with publicly available sequenced genomes have been screened for the presence of sesquiterpene cyclase homologues, resulting in the identification of 55 putative geosmin synthases, 23 homologues of 2-methylisoborneol synthases, and 98 other sesquiterpene cyclase homologues. Most of these enzymes by far were found in actinomycetes. The terpenoid volatiles from 35 strains, including 31 actinomycetes and four strains from other taxa, were collected by using a closed-loop stripping apparatus and identified by GC-MS. All of these bacteria apart from one strain encode sesquiterpene cyclase homologues in their genomes. The identified volatile terpenoids were grouped according to structural similarities and their biosynthetic relationship, and the results of these analyses were correlated to the available genome information, resulting in valuable new insights into bacterial terpene biosynthesis.

Biosynthesis of sesquiterpenes by the fungus Fusarium verticillioides.

The volatiles of the fungus Fusarium verticillioides were analysed by GC-MS. Sesquiterpenes dominated, with trichodiene as the principle component. Several other sesquiterpenes were detected in low amounts that were unambiguously identified from their mass spectra and retention indices. The absolute configurations of (R)-ß-bisabolene, (R)-cuparene, (+)-ß-barbatene, (-)-α-cedrene, (+)-ß-cedrene, and (+)-α-funebrene originating from different key cationic intermediates, were determined by chiral GC-MS and proved to be related to the trichodiene stereostructure. The unusual compound (E)-iso-γ-bisabolene was also found corroborating a previously suggested mechanism for the cyclisation of the bisabolyl to the cuprenyl cation that is based on quantum mechanical calculations (Y. J. Hong, D. J. Tantillo, Org. Lett. 2006, 8, 4601-4604). These analyses resulted in a revised biosynthesis scheme to trichodiene and the side products of the responsible terpene cyclase, trichodiene synthase, an enzyme that is well characterised from Fusarium sporotrichioides. Feeding studies with several deuterated mevalonolactone isotopomers unravelled stereochemical aspects of the late cyclisations towards trichodiene.