Microbial-type terpene synthases significantly contribute to the terpene profile of glandular trichomes of the fern Dryopteris fragrans (L.).

Ferns, known for their adaptability and widespread presence, form a diverse group of plants. However, the mechanisms underlying terpenoid production, which are often linked to plant adaptation, are not well understood in ferns. Here, we report that Dryopteris fragrans (D.

Biosynthesis of the Non-Canonical C Sesquiterpenoids Chlororaphen A and B from Pseudomonas Chlororaphis.

Chlororaphens A and B are structurally unique non-canonical C sesquiterpenoids from Pseudomonas chlororaphis that are made by two SAM-dependent methyltransferases and a type I terpene synthase. This study addresses the mechanism of their formation in isotopic labelling experiments and DFT calculations. The results demonstrate an astonishing complexity with distribution of labellings within a cyclopentane core that is reversely connected to two acyclic fragments in chlororaphen A and B.

Isotopic labelings for mechanistic studies.

The intricate mechanisms in the biosynthesis of terpenes belong to the most challenging problems in natural product chemistry. Methods to address these problems include the structure-based site-directed mutagenesis of terpene synthases, computational approaches, and isotopic labeling experiments. The latter approach has a long tradition in biosynthesis studies and has recently experienced a revival, after genome sequencing enabled rapid access to biosynthetic genes and enzymes.

Common Biosynthesis of Non-Canonical C Terpenes through a Fragmentation-Recombination Mechanism.

The biosynthesis of six recently reported non-canonical C sesquiterpenoids named after ancient Greek philosophers, archimedene, aristotelene, eratosthenene, pythagorene, α-democritene and anaximandrene, was investigated through density functional theory (DFT) calculations and isotopic labeling experiments. The results revealed for all compounds except archimedene a unique fragmentation-recombination mechanism as previously demonstrated for sodorifen biosynthesis, in addition to a remarkable "dancing" mechanism for anaximandrene biosynthesis.

Structural Insights Into the Terpene Cyclization Domains of Two Fungal Sesterterpene Synthases and Enzymatic Engineering for Sesterterpene Diversification.

Little is known about the structures and catalytic mechanisms of sesterterpene synthases (StTSs), which greatly hinders the structure-based engineering of StTSs for structural diversity expansion of sesterterpenes. We here report on the crystal structures of the terpene cyclization (TC) domains of two fungal StTSs: sesterfisherol synthase (NfSS) and sesterbrasiliatriene synthase (PbSS). Both TC structures contain benzyltriethylammonium chloride (BTAC), pyrophosphate (PPi), and magnesium ions (Mg), clearly defining the catalytic active sites.

Functional and Mechanistic Characterization of the 4,5-diepi-Isoishwarane Synthase from the Liverwort Radula lindenbergiana.

The microbial type sesquiterpene synthase RlMTPSL4 from the liverwort Radula lindenbergiana was investigated for its products, showing the formation of several sesquiterpene hydrocarbons. The main product was structurally characterized as the new compound 4,5-diepi-isoishwarane, while the side products included the known hydrocarbons germacrene A, α-selinene, eremophilene and 4,5-diepi-aristolochene. The cyclization mechanism towards 4,5-diepi-isoishwarane catalyzed by RlMTPSL4 was investigated through isotopic labeling experiments, revealing the stereochemical course for the deprotonation step to the neutral intermediate germacrene A, a reprotonation for its further cyclization, and a 1,2-hydride shift along the cascade.

Mechanistic characterisation of a sesquiterpene synthase for asterisca-1,6-diene from the liverwort and implications for pentalenene biosynthesis.

A sesquiterpene synthase from the liverwort was characterised and shown to produce the new sesquiterpene hydrocarbon (3,9)-asterisca-1,6-diene, besides small amounts of pentalenene. The biosynthesis of asterisca-1,6-diene was studied through isotopic labelling experiments, giving additional insights into the long discussed biosynthesis of pentalenene.

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.

Mechanistic Characterisation of the Bacterial Sesterviridene Synthase from Kitasatospora viridis.

A gene coding for a terpene synthase homolog from Kitasatospora viridis was cloned and expressed in Escherichia coli. The purified recombinant protein possessed sesterterpene synthase activity and efficiently converted geranylfarnesyl diphosphate (GFPP) with 19 % yield into the sesterterpene hydrocarbon sesterviridene A. Large scale enzymatic conversions also allowed for the isolation of two side products that are generated with very low yields of ca.

Fragmentation and [4 + 3] cycloaddition in sodorifen biosynthesis.

Terpenes constitute the largest class of natural products. Their skeletons are formed by terpene cyclases (TCs) from acyclic oligoprenyl diphosphates through sophisticated enzymatic conversions. These enzyme reactions start with substrate ionization through diphosphate abstraction, followed by a cascade reaction via cationic intermediates.

Biosynthesis of the Sesquiterpene Kitaviridene through Skeletal Rearrangement with Formation of a Methyl Group Equivalent.

A sesquiterpene synthase from was discovered and shown to produce kitaviridene, a sesquiterpene hydrocarbon with an additional methyl group equivalent in comparison to a regular sesquiterpene. Isotopic labeling experiments together with DFT calculations gave detailed insights into the cyclization cascade toward kitaviridene and explained the formation of the additional methyl group equivalent.

Germacrene B - a central intermediate in sesquiterpene biosynthesis.

Germacranes are important intermediates in the biosynthesis of eudesmane and guaiane sesquiterpenes. After their initial formation from farnesyl diphosphate, these neutral intermediates can become reprotonated for a second cyclisation to reach the bicyclic eudesmane and guaiane skeletons. This review summarises the accumulated knowledge on eudesmane and guaiane sesquiterpene hydrocarbons and alcohols that potentially arise from the achiral sesquiterpene hydrocarbon germacrene B.

A Detailed View on Geosmin Biosynthesis.

The bacterial geosmin synthase is a fascinating bifunctional enzyme that has been discovered almost two decades ago. Several aspects of the cyclisation mechanism from FPP to geosmin are known, but a detailed picture of the stereochemical course of this reaction is unknown. This article reports on a deep investigation of the mechanism of geosmin synthase through isotopic labelling experiments.

Origin of the 3-methylglutaryl moiety in caprazamycin biosynthesis.

Background: Caprazamycins are liponucleoside antibiotics showing bioactivity against Gram-positive bacteria including clinically relevant Mycobacterium tuberculosis by targeting the bacterial MraY-translocase. Their chemical structure contains a unique 3-methylglutaryl moiety which they only share with the closely related liposidomycins. Although the biosynthesis of caprazamycin is understood to some extent, the origin of 3-methylglutaryl-CoA for caprazamycin biosynthesis remains elusive.

Synthesis of tryptophan-dehydrobutyrine diketopiperazine and biological activity of hangtaimycin and its co-metabolites.

An improved synthesis for tryptophan-dehydrobutyrine diketopiperazine (TDD), a co-metabolite of the hybrid polyketide/non-ribosomal peptide hangtaimycin, starting from ʟ-tryptophan is presented. Comparison to TDD isolated from the hangtaimycin producer confirmed its configuration. The X-ray structure of the racemate shows an interesting dimerisation through hydrogen bridges.

Crystal Structure Based Mutagenesis of Cattleyene Synthase Leads to the Generation of Rearranged Polycyclic Diterpenes.

The crystal structures of cattleyene synthase (apo-CyS), and CyS complexed with geranylgeranyl pyrophosphate (GGPP) were solved. The CyS variant exhibited an increased production of cattleyene and other diterpenes with diverse skeletons. Its structure showed a widened active site cavity explaining the relaxed selectivity.

Hedycaryol - Central Intermediates in Sesquiterpene Biosynthesis, Part II.

The known sesquiterpenes that arise biosynthetically from hedycaryol are summarised. Reasonings for the assignments of their absolute configurations are discussed. The analysis provided here suggests that reprotonations at the C1=C10 double bond of hedycaryol are directed toward C1 and generally lead to 6-6 bicyclic compounds, while reprotonations at the C4=C5 double bond occur at C4 and result in 5-7 bicyclic compounds.

The enzyme mechanism of patchoulol synthase.

Different mechanisms for the cyclisation of farnesyl pyrophosphate to patchoulol by the patchoulol synthase are discussed in the literature. They are based on isotopic labelling experiments, but the results from these experiments are contradictory. The present work reports on a reinvestigation of patchoulol biosynthesis by isotopic labelling experiments and computational chemistry.

Isotopic Labeling Experiments Solve the Hedycaryol Problem.

Hedycaryol is a widespread sesquiterpene alcohol and important biosynthetic intermediate toward eudesmols and guaiols. A full NMR assignment for this compound has been hampered because of the unique molecular mechanics of its conformers in complex mixtures. This problem was solved through the enzymatic synthesis of isotopically labeled materials using a mutated plant and a bacterial enzyme for access to both enantiomers of hedycaryol, which also allowed us to follow the stereochemical course of its Cope rearrangement.

Stereochemical characterisation of the non-canonical α-humulene synthase from .

The non-canonical fungal α-humulene synthase was investigated through isotopic labelling experiments for its stereochemical course regarding inversion or retention at C-1, the face selectivity at C-11, and the stereoselectivity of the final deprotonation. A new and convenient desymmetrisation strategy was developed to enable a full stereochemical analysis of the catalysed steps to the achiral α-humulene product from stereoselectively labelled farnesyl diphosphate.

The Mechanism of Dehydrating Bimodules in trans-Acyltransferase Polyketide Biosynthesis: A Showcase Study on Hepatoprotective Hangtaimycin.

A bioassay-guided fractionation led to the isolation of hangtaimycin (HTM) from Streptomyces spectabilis CCTCC M2017417 and the discovery of its hepatoprotective properties. Structure elucidation by NMR suggested the need for a structural revision. A putative HTM degradation product was also isolated and its structure was confirmed by total synthesis.

1,2- or 1,3-Hydride Shifts: What Controls Guaiane Biosynthesis?

A systematic computational study addressing the entire chemical space of guaianes in conjunction with an analysis of all known compounds shows that 1,3-hydride shifts are rare events in guaiane biosynthesis. As demonstrated here, 1,3-hydride shifts towards guaianes can only be realized for two stereochemically well defined out of numerous possible stereoisomeric skeletons. One example is given by the mechanism of guaia-4(15)-en-11-ol synthase from California poplar, an enzyme that yields guaianes with unusual stereochemical properties.