Christian A. Citron

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.

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.

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.

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.

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)‐β‐bisabolene, (R)‐cuparene, (+)‐β‐barbatene, (−)‐α‐cedrene, (+)‐β‐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.

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.

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

Summary Members of the marine Roseobacter clade can degrade dimethylsulfoniopropionate (DMSP) via competing pathways releasing either methanethiol (MeSH) or dimethyl sulfide (DMS). Deuterium-labeled [2H6]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 [2H6]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 [2H3]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 [2H3]methionine and 34SO4 2−, synthesized from elemental 34S8, 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.

Biosynthesis of acorane sesquiterpenes by Trichoderma

The volatiles of three Trichoderma strains have been collected by use of a closed-loop stripping apparatus (CLSA) and analysed by GC-MS. Several biosynthetically related sesquiterpenes have been identified in the complex headspace extracts. Additionally, the absolute configurations of some sesquiterpenes were determined by chiral GC-MS. The biosynthesis of the main compound tricho-acorenol was investigated by feeding of deuterated mevalonolactone isotopomers and contradicts a previously published pathway for Fusidium.